Identification of epigenetic signatures indicating breast cancer

ABSTRACT

In various aspects and embodiments, the invention provides a method of determining breast cancer status of a subject, the method comprising determining a methylation state for each of a plurality of cytosine-guanine dinucleotide (CpG) sites in a sample obtained from the subject, calculating a cancer presence differential methylation level and an invasiveness differential methylation level based on the methylation states of the plurality of CpG sites, and comparing the cancer presence differential methylation level and the invasiveness differential methylation level to a predetermined cancer status reference level and a predetermined invasiveness reference level, wherein when the cancer presence differential methylation level deviates from the predetermined cancer status reference level, the presence of breast cancer is indicated in the subject, and when the invasiveness differential methylation level deviates from the predetermined invasiveness reference level, the presence of invasive breast cancer is indicated in the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/558,124, filed Sep. 13, 2017,which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Breast cancer is the most commonly diagnosed cancer in women worldwide.Although the intent of increased mammographic screening is earlydetection of invasive cancer, an unexpected consequence has been asubstantial increase in the number of women diagnosed with ductalcarcinoma in situ (DCIS). DCIS is considered a benign lesion thatconsists of non-invasive neoplastic cells. Although DCIS has beenconsidered a precursor lesion to invasive breast cancers, only a smallpercentage of DCIS cases progress to invasive cancers. The most commontreatment for DCIS is breast-conserving surgery followed by radiation.Recently, there has been considerable debate over the treatment of DCIS,with many claiming that current treatment regimens result inovertreatment of a mostly indolent disease.

While some groups recommend frequent follow up and monitoring of womenwith low-grade DCIS instead of invasive treatment, this approach wouldnecessitate either frequent biopsy, an unattractive option for mostwomen, or additional mammography. Additionally, for some women,mammography may be hard to interpret or may provide a false negative. Inparticular, dense stromal and epithelial tissue within the breast maycause high background and women with high breast density are more atrisk for a false-negative mammogram. Likewise, certain forms of breastcancer such as triple-negative breast cancer (TNBC) are less likely tobe detected by mammographic screening. Although TNBC may have largersize at diagnosis compared with other breast cancer subtypes, up to 18%of TNBCs remain unidentified on initial screening. This is due to thefact TNBC lacks the typical suspicious mammographic features of breastcancer, such as irregular mass shape, spiculated margins, associatedductal carcinoma in situ and suspicious calcifications. Thus mammographyalone may be a suboptimal test for diagnosis of TNBC, especially forthose at high risk.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of determining breastcancer status of a subject, the method comprising:

determining a methylation state for each of a plurality ofcytosine-guanine dinucleotide (CpG) sites in a sample obtained from thesubject,

calculating a cancer presence differential methylation level and aninvasiveness differential methylation level based on the methylationstates of the plurality of CpG sites, and

comparing the cancer presence differential methylation level and theinvasiveness differential methylation level to a predetermined cancerstatus reference level and a predetermined invasiveness reference level,

wherein when the cancer presence differential methylation level deviatesfrom the predetermined cancer status reference level, the presence ofbreast cancer is indicated in the subject, and

when the invasiveness differential methylation level deviates from thepredetermined invasiveness reference level, the presence of invasivebreast cancer is indicated in the subject.

In another aspect, the invention provides a method of detecting breastcancer in a subject, the method comprising:

determining a methylation state for each of a plurality ofcytosine-guanine dinucleotide (CpG) sites in a sample obtained from thesubject,

calculating a cancer status differential methylation level based on themethylation states of the plurality of CpG sites, and

comparing the cancer status reference differential methylation level toa predetermined reference level,

wherein when the cancer status differential methylation level deviatesfrom the predetermined reference level, the presence of breast cancer isindicated in the subject.

In another aspect, the invention provides a method of determining ifbreast cancer in a subject is invasive, or non-invasive, the methodcomprising:

determining a methylation state for each of a plurality ofcytosine-guanine dinucleotide (CpG) sites in a sample obtained from thesubject,

calculating an invasiveness differential methylation level based on themethylation states of the plurality of CpG sites, and

comparing the invasiveness differential methylation level to apredetermined reference level,

wherein when the differential methylation level deviates from thepredetermined reference level, the breast cancer in the subject isinvasive.

In various embodiments, the plurality of CpG sites comprises at leastone selected from the CpG sites listed in Table 3 or Table 15.

In various embodiments, the plurality of CpG sites comprises at leastfive selected from the CpG sites listed in Table 21.

In various embodiments, the plurality of CpG sites comprises at leastten selected from the CpG sites listed in Table 3 or Table 15.

In various embodiments, the plurality of CpG sites comprises at leastten selected from the CpG sites listed in Table 21.

In various embodiments, the plurality of CpG sites comprises at least m% selected from the top n most predictive CpG sites listed in Table 3and/or Table 15, wherein:

m is selected from the group consisting of: 50, 60, 70, 80, 90, 95, and99; and

n is selected from the group consisting of 25, 50, 100, 500 and 1,000.

In various embodiments, the plurality of CpG sites comprises at least m% selected from the top n most predictive CpG sites listed in Table 21,wherein:

m is selected from the group consisting of: 50, 60, 70, 80, 90, 95, and99; and

n is selected from the group consisting of 25, 50, 100, 500 and 1,000.

In various embodiments, the method further comprises providing treatmentfor breast cancer to the subject when breast cancer is indicated.

In various embodiments, the treatment for breast cancer comprises theadministration of medication, radiation or surgery.

In various embodiments, calculating a differential methylation levelcomprises adding in a linear weighted summation values based on themethylation states of the plurality of CpG sites.

In various embodiments, the sample is a blood sample.

In various embodiments, the sample is tumor tissue.

In various embodiments, the subject has or is suspected to have ductalcell in situ carcinoma.

In various embodiments, the subject has or is suspected to havetriple-negative breast cancer.

In various embodiments, the subject has or is suspected to have hormonereceptor positive (ER+PR+) breast cancer.

In various embodiments, the subject has or is suspected to have HER2+breast cancer.

In various embodiments, the subject is being monitored for the local orsystemic recurrence of breast cancer.

In various embodiments, the plurality of CpG sites includes at least oneselected from table 27.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and desired objects of thepresent invention, reference is made to the following detaileddescription taken in conjunction with the accompanying figures.

FIG. 1A depicts % methylation frequency profiles in individual samplesfrom the control, invasive and DCIS subject groups.

FIG. 1B depicts a comparison of differential methylation load by genefunctional class and domain structure for control and DCIS subjectgroups.

FIG. 1C depicts a non-metric multidimensional scaling analysis toidentify discriminating 5mC patterns among all three subject groups.

FIG. 1D depicts the distribution of the strength vectors of CpG sitescontributing to the group separation in the NMDS plot depicted in FIG.1C.

FIGS. 2A-C depicts CpG methylation as a heatmap for the top 1,000statistically significant sites based on a Likelihood-Ratio-Test (LRT)of a one-way ANOVA contrast for the three-way analysis. Table 8 containsthe counts of the top 40 statistically significant sites.

FIG. 2D depicts a hierarchical clustering of methylation patterns amongtop CpG sites to identify those sites or domains with correlated shiftsin methylation.

FIGS. 3A-C depict CpG methylation distribution from LRT in FIG. 2A-C,respectively.

FIGS. 3D-F depict CpG fold-change vs p-val in volcano plots from LRT inFIG. 2A-C, respectively.

FIG. 4 depicts methylation load across the genome.

FIG. 5A depicts % methylation frequency profiles in individual samplesfrom the control and DCIS subject groups.

FIG. 5B depicts a comparison of differential methylation load by genefunctional class and domain structure for control and DCIS subjectgroups.

FIG. 5C depicts a non-metric multidimensional scaling analysis toidentify discriminating 5mC patterns among control and DCIS subjectgroups.

FIG. 5D depicts the distribution of the strength vectors of CpG sitescontributing to the group separation in the NMDS plot depicted in FIG.5C.

FIG. 6A depicts CpG methylation as a heatmap for the top 1,000statistically significant sites based on a Likelihood-Ratio-Test of aone-way ANOVA contrast for the control and DCIS subject groups. Table 14contains the counts of the top 40 statistically significant sites.

FIG. 6B depicts a hierarchical clustering of methylation patterns amongtop CpG sites to identify those sites or domains with correlated shiftsin methylation.

FIG. 7A depicts CpG response distribution in smear plots from LRT inFIG. 6A.

FIG. 7B depicts CpG response distribution in volcano plots from LRT inFIG. 6A. depicts a heatmap clustering of the top 1,000 CpG sites rankedby p-value.

FIG. 7C depicts methylation load across the genome.

FIG. 8A depicts % methylation frequency profiles in individual samplesfrom the control and invasive breast cancer subject groups.

FIG. 8B depicts a comparison of differential methylation load by genefunctional class and domain structure for control and invasive breastcancer subject groups.

FIG. 8C depicts non-metric multidimensional scaling analysis to identifydiscriminating 5mC patterns among control and invasive breast cancersubject groups.

FIG. 8D depicts the distribution of the strength vectors of CpG sitescontributing to the group separation in the NMDS plot depicted in FIG.8C.

FIG. 9A depicts CpG methylation as a heatmap for the top 1,000statistically significant sites based on a Likelihood-Ratio-Test of aone way ANOVA contrast for the control and invasive breast cancersubject groups. Table 20 contains the counts of the top 40 statisticallysignificant sites.

FIG. 9B depicts a hierarchical clustering of methylation patterns amongtop CpG sites to identify those sites or domains with correlated shiftsin methylation.

FIG. 10A depicts CpG response distribution in smear plots from LRT inFIG. 9A.

FIG. 10B depicts CpG response distribution in volcano plots from LRT inFIG. 9A.

FIG. 10C depicts methylation load across the genome.

FIG. 11A depicts % methylation frequency profiles in individual samplesfor the DCIS and invasive breast cancer subject groups.

FIG. 11B depicts a comparison of differential methylation load by genefunctional class and domain structure for DCIS and invasive breastcancer subject groups.

FIG. 11C depicts a non-metric multidimensional scaling analysis toidentify discriminating 5mC patterns between DCIS and invasive breastcancer subject groups.

FIG. 11D depicts distribution of the strength vectors of CpG sitescontributing to the group separation in the NMDS plot in FIG. 11C.

FIG. 12A depicts CpG methylation as a heatmap for the top 1,000statistically significant sites based on a Likelihood-Ratio-Test of aone way ANOVA contrast for the DCIS and invasive breast cancer subjectgroups. Table 26 contains the counts of the top 40 statisticallysignificant CpG sites.

FIG. 12B depicts a hierarchical clustering of methylation patterns amongtop CpG sites to identify those sites or domains with correlated shiftsin methylation.

FIG. 13A depicts CpG response distribution in smear plots from LRT inFIG. 12A.

FIG. 13B depicts CpG response distribution in volcano plots from LRT inFIG. 12A.

FIG. 13C depicts methylation load across the genome.

FIG. 14 is an NMDS plot for the replicate cohort used to show theseparation on blood DNA methylation profiles between healthy women,women with advanced breast cancer (“invasive”), and women with DCISlesions.

FIG. 15 is a heat map of top 1,000 statistically significant CpG siteswhen comparing CON and INV patient groups. These CpG sites andmethylation load scores for CON and INV patients were used in theclassification platform.

FIG. 16 depicts pie charts presenting the classification calls for the10 blind samples. The platform uses a voting/polling approach and so thepie charts present the proportion of votes that each sample received foreach phenotype class (CON or INV). Decisions were made based on simplemajority counts.

DEFINITIONS

The instant invention is most clearly understood with reference to thefollowing definitions.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

As used herein, the term “breast cancer” refers to pre-malignant ormalignant tumors that start in the epithelial cells of the breast.Breast cancer can be further defined into pathological subtypes based onexpression of the estrogen, progesterone, and HER2 receptors.Additionally, breast cancer can be defined based on molecular subtype asdefined, for example, by Peru et al, Nature. 2000 Aug. 17;406(6797):747-5. Molecular subtypes include luminal A, B, and C, HER 2,basal-like, and normal type.

As used in the specification and claims, the terms “comprises,”“comprising,” “containing,” “having,” and the like can have the meaningascribed to them in U.S. patent law and can mean “includes,”“including,” and the like.

As used herein, “cytosine-guanine dinucleotide site” or “CpG site” meansa cytosine nucleotide followed by a guanine nucleotide in the genome ofan organism. CpG sites can be designated with the number of thechromosome of the organism on which they are located and a numberdesignating the position. The flanking sequences can be used to generatethe position number. For example, “12.108079458” or “chr12 108079458”refer to a CpG site on chromosome 12 at position 108079458. The positionnumber refers to the nucleotide index starting from 1 on the coding orplus (+) strand of the DNA molecule and specifically references theposition of the 5′ cytosine in a CpG dinucleotide pair. In addition,this CpG location has a complementary sequence pair on the non-codingminus (−) strand and the position number also refers to thatcomplementary strand cytosine which is located plus one nucleotide fromthe indicated coding strand position. Thus, for CpG 12.108079458, themethylation score values indexed to this specific site cover thecytosine on the coding strand of chromosome 12 at position number108079458 and the cytosine on the non-coding strand of chromosome 12 atposition number 108079459.

As used herein, the term “carcinoma in situ” refers to a neoplasticlesion characterized by increased epithelial proliferation, subtle tomarked cellular atypia and an inherent but not necessarily obligatetendency for progression to invasive breast cancer as defined by LakhaniS, Ellis I, Schnitt S, et al. 4th. Lyon: IARC Press; 2012. WHOClassification of Tumours of the Breast. Lobular carcinoma in situ(LCIS) refers to a lesion originating from the terminal ductal lobularunits, whereas, ductal carcinoma in situ (DCIS) refers to anintraductoral lesion. DCIS is also known as intraductal carcinoma,ductal intraepithelial neoplasia and includes the following subtypescribiform, solid, comedo, papillary, and micropapilary. DCIS can also becharacterized as low, moderate and high grade.

As used herein, the term “invasive breast cancer” means a malignantepithelial tumor of the breast, characterized by invasion of adjacenttissues and a marked tendency to metastasize to distant sites as definedby Lakhani S, Ellis I, Schnitt S, et al. 4th. Lyon: IARC Press; 2012.WHO Classification of Tumours of the Breast. As used herein, the termencompasses both cancer that presently exhibits these qualities andcancer that will develop them over the course of disease progression.Invasive breast cancer includes the following subtypes: invasivecarcinoma of no special subtype, invasive carcinoma of mixed type,invasive ductal carcinoma, invasive lobular carcinoma, infiltratingcarcinoma of the breast, tubular carcinoma, invasive cribriformcarcinoma, carcinoma with medullary features, mucinous carcinoma,invasive papillary carcinoma, inflammatory breast cancer, Paget diseaseof the breast, metaplastic breast cancer, carcinomas with aprocrinedifferentiation, adenoid cystic carcinoma, microinvasive carcinoma, andcarcinoma with neuroendrocrine features.

As used herein, “hormone receptor positive breast cancer” and “HER2+”mean cancers which have positive expression of estrogen or progesteronereceptors in greater than 1-9% percent of cells. Hormone receptorpositive tumors may or may not have overexpression of the HER2 receptornoted either by immunohistochemical or genetic evaluation.

As used herein, “methylation” or “methylated” as applied to CpG sitesrefers to the addition of a methyl group to cytosine, forming either5′-methyl-cytosine or 5′-hydroxymethyl-cystosine.

As used herein, the term “percent methylation” or “% MET” refers to thefrequency with which a particular set of CpG sites are methylated. Here,CpG methylation is expressed as a percentage of methylated copies foundin the DNA sample for each individual CpG site relative to the totalnumber of copies found for each site.

A “reference level” with respect to some measurement used in diagnosisis indicative of the presence or absence of a particular phenotype orcharacteristic. When the level of the measurement in a subject deviatesfrom the reference level it is indicative of the presence of, orrelatively heightened level of, a particular phenotype orcharacteristic.

As used herein, the term “triple negative breast cancer” means breastcancer in which less than 1-9% of cells express the estrogen orprogesterone receptors and there is no alteration of the HER2 receptornoted either by immunohistochemical or genetic evaluation. The majorityof triple negative breast cancer have gene expression profiles that arerepresentative of the basal subtype of breast cancer.

Unless specifically stated or obvious from context, the term “or,” asused herein, is understood to be inclusive.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (aswell as fractions thereof unless the context clearly dictatesotherwise).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention provides a method of determining the breastcancer status of a subject by determining a methylation state for eachof a plurality of cytosine-guanine dinucleotide (CpG) sites in a sampleobtained from the subject, calculating a cancer status differentialmethylation level and an invasiveness differential methylation levelbased on the methylation states of the plurality of CpG sites, andcomparing the cancer presence differential methylation level and theinvasiveness differential methylation level to a predetermined cancerstatus reference level and a predetermined invasiveness reference level,wherein when the cancer status differential methylation level deviatesfrom the predetermined cancer status reference level, the presence ofbreast cancer is indicated in the subject, and when the invasivenessdifferential methylation level deviates from the predeterminedinvasiveness reference level, the presence of invasive breast cancer isindicated in the subject. This aspect of the invention is based in parton the three-way analysis illustrated in FIGS. 1-4 and discussed furtherin the example below.

Aspects of the invention may be applied to determine if a patient isfree from breast cancer, has DCIS or has invasive breast cancer. Thismay be achieved by determining if the patient's epigenetic profile,based on a plurality of CpG sites and expressed as a cancer presencedifferential methylation level and an invasiveness differentialmethylation level, deviates from the epigenetic profile of patientswithout breast cancer or with DCIS, expressed as a cancer presencereference level and invasiveness reference level. When the patient'scancer presence differential methylation level deviates from the cancerpresence reference level, the presence of cancer (invasive or not) isindicated. When the patient's invasiveness differential methylationlevel, deviates from the invasiveness reference level, the presence ofinvasive breast cancer is indicated. In various embodiments, thesubjects may have invasive breast cancer associated with carcinoma insitu. A person of skill in the art will appreciate that the methods ofthe invention may be used to detect this condition by calculating acarcinoma in situ differential methylation level based on a plurality ofthe CpG sites disclosed herein using the below described methods.

In another aspect, the invention provides a method of detecting breastcancer in a subject by determining a methylation state for each of aplurality of cytosine-guanine dinucleotide (CpG) sites in a sampleobtained from the subject, calculating a cancer status differentialmethylation level based on the methylation states of the plurality ofCpG sites, and comparing the cancer status differential methylationlevel to a predetermined cancer status reference level, wherein when thedifferential methylation level deviates from the predetermined cancerstatus reference level, the presence of breast cancer is indicated inthe subject. The methods of the invention need not be applied todetermine if the cancer is invasive. The method may also be applied todetect cancer by determining if the patient's epigenetic profile, basedon a plurality of CpG sites and expressed as a cancer statusdifferential methylation level, deviates from the epigenetic profile ofpatients without breast cancer, expressed as a cancer status referencelevel. When the patient's cancer status differential methylation leveldeviates from the cancer status reference level, the presence of breastcancer in the patient is indicated.

In another aspect, the invention provides a method of determining ifbreast cancer in a subject is invasive or non-invasive by determining amethylation state for each of a plurality of CpG sites in a sampleobtained from the subject, calculating an invasiveness differentialmethylation level based on the methylation states of the plurality ofCpG sites, and comparing the invasiveness differential methylation levelto a predetermined reference level, wherein when the differentialmethylation level deviates from the predetermined reference level, thepresence of breast cancer is indicated in the subject. This aspect ofthe invention is related to the DCIS vs. invasive analysis illustratedin FIGS. 11-13 and discussed further in the example below.

The methods of the invention may be applied to patients who havepreviously been diagnosed or are otherwise believed to have breastcancer, in order to determine whether or not the cancer is invasive.When the patient's invasiveness differential methylation level deviatesfrom the invasiveness reference level, the presence of invasive breastcancer is indicated.

In another aspect, the invention provides a method of detecting local orsystemic recurrence of breast cancer by determining a methylation statefor each of a plurality of cytosine-guanine dinucleotide (CpG) sites ina sample obtained from the subject, calculating a cancer statusdifferential methylation level and an invasiveness differentialmethylation level based on the methylation states of the plurality ofCpG sites, and comparing the cancer presence differential methylationlevel and the invasiveness differential methylation level to apredetermined cancer status reference level and a predeterminedinvasiveness reference level, wherein when the cancer statusdifferential methylation level deviates from the predetermined cancerstatus reference level, the recurrence of breast cancer is indicated inthe subject, and when the invasiveness differential methylation leveldeviates from the predetermined invasiveness reference level, therecurrence of invasive breast cancer is indicated in the subject.

In various embodiments, the patient has or is suspected to have triplenegative breast cancer. In various embodiments, the patient has or issuspected to have hormone receptor positive (ER+PR+, or ER+PR− or ER−PR+) breast cancer. In various embodiments, the patient has or issuspected to have HER2+ breast cancer.

The methylation of state of the plurality of CpG sites may be determinedby any means known in the art. In various embodiments, the methylationstate of the plurality of CpG sites may be determined bymethyl-sensitive restriction enzyme digestion followed by Next-GenSequencing (NGS) on an appropriate instrument, or they may be determinedby targeted qPCR assays to quantify cut and uncut CpG sites followingmethyl-sensitive restriction enzyme digestion, or they may be determinedby bisulfite oxidation treatment with DNA sequencing (either direct orvia NGS), or they may be determined by hybridization of labelledoligonucleotide probes (called “hybridization arrays”) to measuremethylation following methyl-sensitive restriction enzyme digestion, orthey may be determined by hybridization of anti-5′-methyl-cytosineantibodies to measure methylation after hybridization capture on atargeted gene panel.

Without wishing to be limited by theory, in various embodiments, themethod relies on the concept of differential methylation level(ΔML)—site-specific differences in CpG methylation summed across a geneor genome domain, structure or element—in order to characterizefunctional shifts in methylation patterns. This method is illustrated inthe example below and in Equation (1). In various embodiments,calculating a differential methylation level comprises adding in alinear weighted summation values based on the methylation states of theplurality of CpG sites.

As shown in FIGS. 1D, 5D, 8D, and 11D, the methylation state of themajority of CpG sites in the genome are not significant predictors ofbreast cancer in a patient. However, determining the methylation stateof a certain subset of CpG sites and applying the algorithm in theexample below or a derivative classification algorithm, one may be usedto determine the breast cancer status of a patient. The plurality of CpGsites in the present aspect of the invention refers to sites selectedfrom this predictive subset of sites. A list of predictive sites andtheir predictive power as measured by p-value and ordinatediscrimination is presented in the Appendix.

In various embodiments, the plurality of CpG sites includes a pluralityof “up sites” and a plurality of “down sites”. Up sites are CpG siteswhere methylation at the site indicates an increased methylation load inpatients in the first group in the comparison relative to patients inthe second group in the comparison. Down sites are CpG sites wheremethylation at the site indicates a decreased methylation load in thefirst group in the comparison relative to patients in the second groupin the comparison.

Various embodiments of the invention are directed to methods ofexamining sets of up sites and down sites and determining a differentialmethylation level based on their methylation state. Due to thepredictive value of the plurality of sites, the scores will differ whenexamining patients with and without breast cancer and where the breastcancer is invasive or not.

The various reference levels in the above-described aspects may becalculated by determining the methylation state of a set of sites inpatients who are known not to have breast cancer or are known to haveDCIS, as appropriate for the respective embodiments. Accordingly, when adifferential methylation level is determined using the same plurality ofsites, deviation from the reference level indicates an additionalevidentiary datum point supporting increasing the probability that thepatient likely has breast cancer or invasive breast cancer.

A skilled person will understand that the specifics of the calculationused for generating a differential methylation level are not criticaland various processes may be employed to generate these levels. All ofthem are within the scope of various embodiments of the invention.

Various embodiments of the invention rely on pluralities of CpG sites ofvarious sizes. In various embodiments, the plurality of CpG sites maycontain about 5, 10, 100, 1000, 5000 or more CpG sites.

Even among the CpG sites that have shown predictive power, differentsites contribute different weightings to the overall predictiveprobability of the cancer status of a patient. Various embodiments ofthe invention calculate differential methylation levels based on variouscombinations of predictive CpG sites.

The predictive power of the sites may be quantified in various ways. Asshown in FIGS. 1C, 5C, 8C and 11C, the deviation between the referencelevel and the differential methylation level for patients with DCIS orinvasive breast cancer can be represented by non-metric multidimensionalscaling (NMDS). Each site in the set will make a contribution to theordinate discrimination in the NMDS. In some embodiments, the pluralityof CpG sites includes one or more sites selected from Tables 3, 9, 15 or21, in which the sites are ranked by the site's contribution to ordinatediscrimination, as appropriate for the various embodiments.

The predictive power of CpG sites may also be quantified based onP-value, adjusted for False Discovery Rate (FDR). Tables 8, 14, 20 and26 list the top 40 CpG sites by P-value. In various embodiments, theplurality of CpG sites in any of the above aspects or embodiments mayinclude one or more sites selected from the Tables below, in particular,those that rank CpG sites by their contribution to NMDS or by p-value.In some embodiments, the plurality of CpG sites may include the top nsites or m of the top n sites from these Tables or other ordinal lists(wherein m and n are positive integers). All of the CpG sites recitedherein may in various embodiments be included in the calculation.

In various embodiments, the plurality of CpG sites may include siteswithin genes that are hypermethylated among patients with DCIS relativeto patients without evidence of breast cancer. Table 11 lists CpG sitesaccording to this metric. In various embodiments, the plurality of CpGsites may include sites within genes that are hypermethylated amongpatients with invasive breast cancer relative to patients withoutevidence of breast cancer. Tables 17 lists CpG sites according to thismetric. In various embodiments, the plurality of CpG sites may includesites within genes that are hypermethylated among patients with invasivebreast cancer relative to patients with DCIS. Table 23 lists CpG sitesaccording to this metric.

In various embodiments, the tumor sample may be any sample obtained fromthe patient that contains sufficient DNA such that the methylationstates of the various CpG sites may be determined. In variousembodiments, the sample may be a blood, saliva or tissue sample. Invarious embodiments, the sample may contain tumor cells. In variousembodiments the sample may be a tumor tissue sample. In variousembodiments, the sample may comprise peripheral blood mononuclear cells(PBMCs). In various embodiments, the sample may be enriched to containpredominantly or substantially exclusively one type of cell or tissue,by way of non-limiting example the sample may be processed to containpredominantly or exclusively PBMCs.

In various embodiments, the method further includes providing treatmentfor breast cancer to patients in whom cancer is indicated. Thistreatment will vary for patients with DCIS or invasive breast cancer.The treatment may include any form of standard of care treatment for theform of cancer indicated accepted by the extended medical community.This includes but is not limited to hormonal therapy, targeted therapy,immunotherapy, chemotherapy, radiation or surgery. In variousembodiments, the treatment may be Evista (Raloxifene Hydrochloride)Keoxifene (Raloxifene Hydrochloride), Raloxifene Hydrochloride,Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilizedNanoparticle Formulation), Ado-Trastuzumab Emtansine, Afinitor(Everolimus), Anastrozole, Aredia (Pamidronate Disodium), Arimidex(Anastrozole), Aromasin (Exemestane), Atezolizumab (Tecentriq)Capecitabine, Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan(Cyclophosphamide), Cisplatin, Carboplatin, Docetaxel, DoxorubicinHydrochloride, Ellence (Epirubicin Hydrochloride), EpirubicinHydrochloride, Eribulin Mesylate, Everolimus, Exemestane, 5-FU(Fluorouracil Injection), Fareston (Toremifene), Faslodex (Fulvestrant),Femara (Letrozole), Fluorouracil Injection, Folex (Methotrexate), FolexPFS (Methotrexate), Fulvestrant, Gemcitabine Hydrochloride, Gemzar(Gemcitabine Hydrochloride), Goserelin Acetate, Halaven (EribulinMesylate), Herceptin (Trastuzumab), Ibrance (Palbociclib), Ipilimumab(Yervoy), Ixabepilone, Ixempra (Ixabepilone), Kadcyla (Ado-TrastuzumabEmtansine), Kisqali (Ribociclib), Lapatinib Ditosylate, Letrozole,Megestrol Acetate, Methotrexate, Methotrexate LPF (Methotrexate), Mexate(Methotrexate), Mexate-AQ (Methotrexate), Neosar (Cyclophosphamide),Niraparib (Zejula), Nivolumab (Opdivo), Nolvadex (Tamoxifen Citrate),Olaparib (Lynparza), Paclitaxel, Paclitaxel Albumin-stabilizedNanoparticle Formulation, Palbociclib, Pamidronate Disodium, Perjeta(Pertuzumab), Pembrolizumab (Keytrundra), Pertuzumab, Ribociclib,Rucaparib (Rubraca), Tamoxifen Citrate, Taxol (Paclitaxel), Taxotere(Docetaxel), Thiotepa, Toremifene, Trastuzumab, Tykerb (LapatinibDitosylate), Velban (Vinblastine Sulfate), Velsar (Vinblastine Sulfate),Vinblastine Sulfate, Xeloda (Capecitabine), Zoladex (Goserelin Acetate)and the like. In various embodiments, where carcinoma in situ isindicated, the treatment may include monitoring or in some embodimentsbe limited to monitoring the benign lesion to ensure that it has notprogressed to an invasive form.

Experimental Example

The invention is further described in detail by reference to thefollowing experimental example. This example is provided for purposes ofillustration only, and is not intended to be limiting unless otherwisespecified. Thus, the invention should in no way be construed as beinglimited to the following example, but rather, should be construed toencompass any and all variations which become evident as a result of theteaching provided herein.

Example 1

In order to test the hypothesis that methylation changes in circulatinglymphocytes could discriminate between women with different stages ofbreast cancer and women without cancer, a sensitive platform to identifychanges in specific CpG profiles in blood cells that correspond todisease states in breast cancer was used. Discrimination of methylationprofiles of peripheral blood mononuclear cells (PBMCs) from women withbreast cancer and healthy women is evident.

Overall, at a functional level, patterns of differential methylation canbe traced back to pathways and genes that support hypotheses about thepotential role of DNA methylation in the altered immunological responseto breast cancer. These methylation signals are evident in blood eventhough they convey a distinct tumor activity signature. Even with asmall pilot cohort (8 normal, 6 DCIS and 8 invasive breast cancersamples) epigenetic profiles that discriminate between normal and tumorblood profiles were recovered. As expected, the distribution of signalsin the tumor samples represent the complexity of the disease; however,the patterns in blood cells from healthy women are relativelyoverlapping

ΔML was calculated as the summation of the difference in % MET scoresfor each CpG site within the defined region or structure being scored,averaged by the number of CpG sites present:

$\begin{matrix}{{\Delta \; {ML}} = {\sum\limits_{i = {first}}^{last}\left\lbrack {{CpG}_{{\lbrack i\rbrack}{\lbrack{{Grp}\; 1}\rbrack}} - {CpG}_{{\lbrack i\rbrack}{\lbrack{{Grp}\; 2}\rbrack}}} \right\rbrack}} & (1)\end{matrix}$

where first and last CpG indexes are defined by the gene unit acrosswhich the summation score is being calculated. Thus, positive ΔML valuesindicate more methylation present in Grp1 and negative values indicatemore methylation in Grp2.

The pairwise analysis in Table 1 provides a direct contrast and allowsfor several graphical visualization plots to be generated these arepresented in FIGS. 1-13. FIGS. 1-4 and Tables 3-8 illustrate thedifferences in epigenetic profile between patients with DCIS, patientswith invasive breast cancer and patients without breast cancer (control)in a three-way analysis. FIGS. 5-7 and Tables 9-14 illustrate thedifferences in epigenetic profile between patients without breast cancer(control) and patients with DCIS. FIGS. 8-10 and Tables 15-20 illustratethe differences in epigenetic profile between patients with invasivebreast cancer and control patients. FIGS. 11-13 and Tables 21-26illustrate the differences in epigenetic profile between patients withDCIS and patients with invasive breast cancer.

As described below in Example 2, a blind study was conducted. The CpGsites used in that analysis are listed in Table 27. In variousembodiments, the plurality of CpG sites may include one or more of thesites listed in Table 27.

Table 1 summarizes the group comparisons and the samples associated witheach group.

TABLE 1 Defined Group Comparisons in this Analysis Code Project 1 Grp1Grp2 Samples 04-ConDCIS Healthy DCIS TB215, TB216, TB022, TB023, TB024,TB007, TB068, TB074, TB078 02-ConINV Healthy INVASIVE TB215, TB216,TB022, TB023, TB024, TB189, TB206, TB208, TB209, TB214, TB009, TB014,TB015, TB017 03-DCIS-INV DCIS INVASIVE TB007, TB068, TB074, TB078 TB189,TB206, TB208, TB209, TB214, TB009, TB014, TB015, TB017 * = HealthySamples TB173 and TB190 are not included in these analyses because oftheir extreme departure in profile from the other Healthy samples. Theyare not similar in methylation pattern, just highly divergent atdifferent CpG sites from the other Healthy samples.

Example 2

To test the prognostic ability of epigenetic analysis of circulatinglymphocytes, a blinded study was executed with an additional 30 samples.It is a separate cohort than the existing cohort, but it is areplication of the cohort with an experimental design that allowed forblind testing. The goal of the classification test was to examine bloodsamples from patients recently diagnosed with DCIS and assess whetherthere was a DNA methylation signature that could be indicative of therisk that the observed breast tissue lesion would be likely to becomeinvasive or remain a benign cell type. For comparison, risk wasdetermined based on independent pathologic criteria determined onlesions after surgical resection. Samples were scored as low risk(Cribroform subtype, no necrosis, low mitotic index) or high risk(Comedo or solid subtype, high mitotic index, necrosis).

Nine out of the 10 blind samples were classified correctly in comparisonto known pathologic criteria. To accomplish this, the control patients'and invasive patients' samples (blood DNA methylation profiles) wereused to develop a discriminating classification routine. The hereindisclosed methods were used to generate the statistical and algorithmicassets required to execute the classification calls on new,blind/unknown samples. The results are presented in FIGS. 14-16 andTable 2, below.

TABLE 2 RISK of invasiveness as determined by: DNA Methylation Samplesize Pathologic Profiling (THIS number Pathological Subtype Gradenecrosis (mm) Criteria METHOD) CORRECT B03 cribriform and comedo 3 yes25 HIGH HIGH correct B04 solid and comedo 3 yes 22 HIGH HIGH correct B12cribriformimg 2 no 3 LOW LOW correct B15 solid 3 yes 10 HIGH HIGHcorrect B18 cribriforming and 1 no 15 LOW LOW correct comedo B21 solidand cribriform 3 yes 30 HIGH LOW wrong B24 cribroform and solid 3 yes 20HIGH HIGH correct B27 cribroform, 1 no 2.5 LOW LOW correctmicropapillary B30 cribriform 2 no 9 LOW LOW correct B33 solid andcribriform 2 yes 18 HIGH HIGH correct

Rankings of Sites for the Three-Way Analysis

TABLE 3 Top 1000 CPG Sites. Rank Ordered listing of CpG sitescontributing the most to the ordinate discrimination in the NMDSanalysis. CpG chr21.0009826110 chr7.0056441233 chr4.0190991653chr13.0027295422 chr20.0033104250 chr21.0009826092 chr12.0054089919chr17.0014203257 chr8.0086728497 chr2.0113240609 chr12.0081330338chr17.0019066525 chr6.0139349539 chr11.0027384364 chr6.0126661739chr22.0042915052 chr11.0061451702 chr13.0100612097 chr4.0190991714chr13.0112720793 chr11.0111101254 chr14.0023057582 chr13.0095363025chr16.0029802815 chr18.0019284903 chr3.0181438281 chr22.0021615439chr15.0045409777 chr13.0061989475 chr17.0033814506 chr13.0028364252chr3.0120626543 chr16.0031227255 chr13.0050656618 chr18.0076738111chr3.0033482718 chr12.0095162637 chr15.0094774231 chr16.0054323769chr6.0161065266 chr5.0172671714 chr2.0096810269 chr3.0197392480chr12.0008113542 chr4.0009215375 chr3.0149531388 chr14.0035008827chr20.0021086995 chr14.0069261542 chr12.0113573025 chr5.0017582586chr3.0138656356 chr14.0060976921 chr17.0038716331 chr15.0063334768chr14.0019893202 chr1.0000565262 chr18.0000499464 chr3.0172167013chr20.0056804010 chr12.0002862275 chr11.0016626136 chr9.0115823339chr10.0135479593 chr2.0177014555 chr11.0070508612 chr11.0064684954chr15.0041794125 chr6.0027247636 chr2.0018060121 chr9.0042368701chr17.0061628676 chr18.0046477561 chr14.0101144066 chr12.0130662393chr11.0009635202 chr17.0043129577 chr8.0086556290 chr14.0037126315chr11.0057407074 chr17.0034497913 chr21.0010164209 chr3.0184302159chr20.0055926272 chr18.0013136247 chr13.0033591525 chr14.0037133183chr13.0068862091 chr6.0159572466 chr22.0040440371 chr20.0048553776chr11.0068607299 chr4.0190990174 chr14.0094255732 chr19.0050595864chr17.0057970085 chr16.0047007494 chr6.0010695540 chr11.0089713826chr12.0106978718 chr6.0027248460 chr16.0023653028 chr20.0048730218chr8.0086570466 chr2.0183731397 chr2.0000729785 chr12.0050506409chr6.0002970717 chr22.0032341165 chr22.0023524377 chr22.0037680206chr17.0012693967 chr14.0035026525 chr18.0030349745 chr11.0009635545chr11.0119211657 chr18.0015197800 chr20.0062282831 chr3.0129118333chr15.0032680456 chr20.0020742515 chr11.0003181639 chr6.0159525700chr17.0041322124 chr5.0140767531 chr14.0058712032 chr22.0021213668chr11.0064889237 chr18.0005238890 chr2.0042329510 chr11.0032113404chr9.0099540553 chr12.0114839068 chr11.0043580678 chr11.0091598917chr20.0009496892 chr13.0112723555 chr16.0055358884 chr8.0061430226chr6.0035182463 chr11.0031846870 chr16.0058498594 chr17.0001954986chr2.0091777959 chr13.0096205001 chr22.0031740075 chr5.0042991671chr20.0060942639 chr20.0058515736 chr3.0149689478 chr6.0157744689chr14.0038071393 chr11.0124734304 chr12.0117674458 chr6.0161034075chr3.0038066016 chr21.0038068930 chr17.0066196740 chr17.0059534001chr6.0026157100 chr18.0029523502 chr15.0100107315 chr11.0064948379chr18.0077960570 chr11.0045433229 chr19.0036193143 chr22.0046299680chr10.0003514879 chr12.0113573398 chr6.0034192386 chr6.0028505184chr6.0026020848 chr17.0009548324 chr18.0044774814 chr9.0084561076chr11.0069323971 chr9.0035603644 chr21.0009826075 chr9.0066457951chr15.0101300081 chr3.0069134445 chr17.0036286187 chr12.0005539802chr15.0075401874 chr12.0124339667 chr5.0042949882 chr17.0043250346chr6.0026158708 chr20.0023017832 chr14.0068286569 chr12.0006641855chr11.0046370207 chr2.0071127961 chr13.0112760981 chr6.0028864916chr11.0118088293 chr15.0060297395 chr12.0040014292 chr6.0005999721chr12.0127210936 chr12.0114886102 chr12.0055247863 chr22.0019746577chr11.0124710147 chr5.0077254287 chr11.0066114505 chr12.0057856280chr20.0056785111 chr22.0049764110 chr9.0132647804 chr12.0007798193chr12.0088535308 chr13.0037574863 chr12.0000248545 chr20.0060310202chr5.0092931810 chr16.0001877823 chr14.0064969377 chr12.0072665650chr20.0062708881 chr3.0045837192 chr11.0041259310 chr3.0066023815chr6.0085477669 chr13.0100648209 chr15.0063893260 chr3.0047323602chr3.0129721027 chr11.0010316376 chr12.0045269062 chr21.0045721062chr2.0230421733 chr4.0191007813 chr3.0197391931 chr14.0029235148chr9.0133537741 chr21.0009826226 chr11.0118306730 chr3.0059466375chr11.0071855196 chr3.0157824506 chr20.0040321851 chr22.0032807421chr12.0095267668 chr13.0048612261 chr13.0095366027 chr2.0012857487chr8.0022551030 chr11.0068593346 chr3.0190323321 chr17.0046723834chr17.0043176656 chr3.0180319542 chr18.0020839973 chr16.0021512858chr17.0056410100 chr14.0037130747 chr14.0072980894 chr14.0101925882chr21.0009826146 chr6.0161037536 chr15.0028343785 chr6.0168110077chr11.0047416016 chr12.0053719703 chr2.0055747731 chr20.0060693185chr17.0058678996 chr15.0066547368 chr12.0132671272 chr18.0076481748chr3.0128209966 chr17.0036719559 chr5.0063257095 chr17.0042634436chr15.0070767299 chr3.0155945555 chr12.0108079458 chr12.0064215916chr11.0031824327 chr20.0031034124 chr20.0002645380 chr6.0134210997chr4.0190988866 chr11.0019546541 chr20.0043513977 chr15.0074365266chr17.0042734551 chr22.0050752817 chr11.0044588016 chr19.0050038397chr17.0045728221 chr8.0086570688 chr18.0027861099 chr6.0010381594chr11.0133401937 chr3.0042845188 chr2.0080549750 chrX.0115003962chr17.0075406104 chr14.0021093009 chr5.0171463182 chr3.0126006910chr14.0097378133 chr22.0050742674 chr15.0089922792 chr12.0050616434chr20.0043595994 chr14.0089290312 chr2.0131356252 chr11.0118794762chr19.0042069923 chr2.0038978853 chr22.0039436523 chr20.0019984304chr11.0001332393 chr3.0160939899 chr13.0022246503 chr12.0041720681chr13.0112708614 chr2.0048648149 chr2.0087304475 chr12.0094361472chr16.0030886974 chr22.0048541823 chr16.0001822579 chr13.0111766619chr3.0126259929 chr11.0001474987 chr9.0110400086 chr7.0121048611chr5.0151151794 chr2.0074056574 chr5.0140782367 chr17.0042072437chr11.0000415553 chr14.0050101778 chr9.0134954595 chr17.0067577554chr18.0057357804 chr16.0030671910 chr6.0027777950 chr5.0054527329chr18.0055096263 chr12.0048358151 chr14.0069658819 chr20.0039995809chr11.0047160760 chr11.0007695679 chr19.0016438474 chr11.0043604860chr17.0056596237 chr20.0058090801 chr11.0134341441 chr18.0007371020chr13.0021715169 chr13.0112330267 chr3.0049044952 chr12.0050453817chr16.0089181384 chr20.0002853406 chr11.0031838687 chr18.0076737080chr12.0014927550 chr6.0150184172 chr15.0101061098 chr6.0136571978chr6.0011537594 chr11.0075111078 chr2.0127415137 chr20.0031330621chr14.0065689243 chr13.0036052554 chr12.0096428593 chr19.0013267022chr7.0024613735 chr3.0127634119 chr5.0003103410 chr17.0017654366chr12.0046783625 chr9.0023824650 chr11.0096072761 chr12.0113313505chr3.0122747539 chr12.0122675633 chr5.0115297957 chr18.0047721730chr5.0069207752 chr15.0048625023 chr17.0059487528 chr12.0010874823chr15.0023439042 chr12.0057630834 chr3.0137484002 chr15.0029864144chr19.0059083815 chr3.0137480414 chr17.0079455749 chr3.0152552924chr2.0020189294 chr15.0028344150 chr11.0067120835 chr3.0119422009chr12.0048397033 chr6.0168079818 chr10.0135480431 chr20.0021083517chr12.0050297405 chr22.0037663127 chr20.0021684369 chr13.0112760512chr11.0104963181 chr2.0095691796 chr17.0080807535 chr16.0012667646chr19.0042498208 chr15.0090743890 chr12.0000863710 chr3.0058554460chr5.0013988181 chr5.0036745487 chr5.0174027285 chr20.0031352450chr2.0217363364 chr11.0060929203 chr22.0017518146 chr18.0049868315chr12.0103696281 chr6.0040995408 chr16.0000766221 chr12.0032908207chr12.0054357015 chr11.0031821274 chr17.0018992459 chr19.0037761411chr18.0046475810 chr12.0070637151 chr11.0019546133 chr6.0126101636chr2.0204975304 chr5.0175960713 chr18.0013562830 chr17.0021023063chr6.0131457089 chr3.0024537205 chr20.0031261476 chr17.0080292993chr3.0008811437 chr22.0037663526 chr16.0030366829 chr14.0024564132chr2.0016081660 chr2.0176946724 chr8.0065281465 chr11.0095657585chr17.0008925911 chr15.0067814061 chr3.0071113928 chr12.0056223792chr17.0046675549 chr11.0082611377 chr21.0009826287 chr15.0063335445chr3.0188040907 chr12.0121018541 chr21.0011143747 chr5.0174155579chr15.0068126000 chr3.0185977002 chr22.0020068641 chr12.0121564042chr2.0045029060 chr5.0007851285 chr1.0000566317 chr13.0099064192chr2.0039665281 chr6.0107386268 chr3.0119813692 chr21.0046130036chr20.0061450707 chr17.0017876238 chr11.0124629895 chr15.0045927689chr11.0046938767 chr17.0036287487 chr17.0001163539 chr15.0041794568chr11.0066187812 chr3.0051704271 chr20.0032320496 chr20.0021690018chr6.0010382800 chr20.0002821334 chr3.0107151212 chr11.0020619810chr18.0074963218 chr17.0007465780 chr16.0057337873 chr15.0099559060chr7.0096653354 chr20.0033542941 chr22.0042467277 chr3.0179182123chr9.0000978453 chr3.0067705228 chr3.0195919117 chr15.0053076316chr15.0089157895 chr2.0068695071 chr15.0066547713 chr12.0072332759chr11.0017553236 chr19.0019976991 chr12.0097700243 chr11.0010229941chr3.0009792106 chr20.0009049993 chr6.0001391144 chr18.0024131895chr17.0048105091 chr12.0075724194 chr15.0072941453 chr2.0133426687chr20.0062486416 chr11.0045101893 chr14.0024615469 chr17.0005342572chr11.0116064679 chr13.0112187940 chr2.0039665069 chr7.0149917678chr20.0003657392 chr11.0118087906 chr12.0004383507 chr7.0139184793chr6.0144606507 chr22.0042678985 chr6.0031409291 chr12.0006450539chr17.0046827033 chr15.0062359382 chr18.0060264186 chr15.0040799671chr6.0027870991 chr18.0077304941 chr5.0037837160 chr15.0040886106chr2.0070315802 chr11.0044327524 chr14.0069950620 chr3.0150805072chr7.0087230305 chr13.0113295221 chr11.0067173421 chr7.0035301934chr7.0121951055 chr21.0038740566 chr6.0027806422 chr5.0006745890chr14.0037074363 chr15.0020774586 chr3.0147128690 chr15.0041221090chr15.0089953564 chr17.0028257894 chr11.0002554562 chr17.0017696370chr17.0073029804 chr5.0075698439 chr6.0151816054 chr2.0114262064chr13.0098312933 chr6.0010694842 chr3.0111632475 chr6.0030313321chr6.0100896338 chr11.0031827853 chr5.0141488834 chr20.0031126668chr18.0011163944 chr15.0033487369 chr11.0070601917 chr20.0057227608chr2.0043450965 chr3.0066025583 chr19.0045931076 chr12.0114877437chr5.0058571658 chr17.0073268308 chr3.0147122989 chr17.0034748982chr20.0034286460 chr12.0007055586 chr15.0075119454 chr5.0124536152chr20.0039312811 chr9.0123555820 chr6.0053214211 chr8.0086727439chr15.0045695003 chr2.0073152645 chr11.0006337183 chr6.0029691943chr8.0008726877 chr12.0065066843 chr15.0068599199 chr18.0040051779chr12.0133135361 chr2.0055459714 chr20.0002854843 chr15.0081475686chr11.0125366224 chr12.0058088019 chr11.0065264490 chr9.0000972268chr15.0040212392 chr15.0026965921 chr6.0071122748 chr11.0001913079chr11.0117685841 chr17.0050236261 chr2.0171569107 chr17.0046655096chr5.0077269177 chr17.0073106082 chr16.0066987650 chr14.0029255068chr11.0100997702 chr6.0161034892 chr22.0019843427 chr3.0075471437chr18.0011639297 chr2.0174831063 chr3.0192959361 chr14.0075077922chr19.0039694617 chr22.0024952038 chr11.0124669378 chr19.0055553285chr11.0001680823 chr20.0033064257 chr20.0029551109 chr15.0043415444chr11.0031979880 chr20.0046601108 chr17.0042393959 chr17.0046696054chr12.0121079347 chr22.0031740052 chr12.0124418736 chr11.0123008747chr7.0039015708 chr3.0185912172 chr18.0045972953 chr16.0018043462chr11.0064085532 chr20.0043514818 chr12.0122377340 chr20.0035274789chr12.0002161319 chr17.0007590759 chr7.0005602782 chr20.0061554910chr13.0112187284 chr19.0012163533 chr17.0073031426 chr12.0057506153chr11.0108368623 chr15.0045410154 chr20.0021284593 chr14.0078227825chr11.0000627511 chr3.0179753765 chr2.0239028866 chr13.0041364285chr2.0148283585 chr12.0008087443 chr3.0014731336 chr18.0022126423chr6.0085482109 chr17.0073629082 chr10.0104962224 chr11.0063687223chr3.0061236525 chr14.0097050980 chr2.0177022661 chr12.0004227877chr2.0232791921 chr12.0067663301 chr11.0093277585 chr2.0220361194chr5.0137668011 chr22.0042487342 chr15.0099193744 chr12.0057118543chr19.0054345439 chr5.0161495517 chr17.0007590939 chr12.0064233302chr2.0120980577 chr6.0163837639 chr17.0033759787 chr17.0004047168chr2.0045241408 chr5.0076010444 chr17.0019883369 chr17.0001000224chr14.0091225099 chr13.0053030043 chr6.0036842708 chr13.0036045456chr12.0006976124 chr12.0052626846 chr3.0136470077 chr12.0056323383chr19.0046312992 chr6.0168842694 chr17.0019088754 chr13.0079018857chr15.0058158361 chr20.0031128317 chr11.0065189464 chr20.0039319920chr7.0027223194 chr8.0080740825 chr20.0042136963 chr5.0042949497chr17.0016284098 chr6.0150284688 chr17.0018996904 chr20.0036661494chr16.0030066605 chr21.0034603400 chr12.0110338921 chr11.0000308473chr5.0088180118 chr11.0130319714 chr16.0005006250 chr18.0012884805chr20.0022549081 chr12.0052414199 chr13.0041496017 chr13.0110761557chr12.0054371989 chr15.0075495213 chr11.0070442560 chr14.0038678690chr19.0000405313 chr18.0055104229 chr14.0090798926 chr17.0007190047chr20.0003389329 chr3.0126195349 chr17.0057915773 chr7.0063361617chr12.0067782441 chr12.0051476565 chr11.0065308645 chr14.0024611126chr13.0112548733 chr4.0190943644 chr3.0107308392 chr11.0093707469chr15.0047477411 chr16.0004364184 chr6.0026033650 chr20.0000591221chr3.0193857514 chr7.0023507247 chr2.0010975677 chr12.0053732109chr6.0157009112 chr19.0044576521 chr3.0024630223 chr20.0062609849chr20.0061584448 chr16.0000216561 chr11.0100558372 chr22.0033040841chr3.0157217384 chr9.0090256959 chr10.0047083620 chr5.0052286121chr22.0037696640 chr9.0005339513 chr12.0056652330 chr3.0120278301chr13.0077014601 chr11.0000382209 chr2.0202126440 chr15.0062599263chr2.0192710945 chr2.0142523231 chr17.0008339890 chr19.0018060689chr11.0002322072 chr16.0032211464 chr10.0103542667 chr5.0159849010chr3.0006904601 chr12.0113489957 chr2.0046727530 chr11.0001676083chr6.0010412348 chr15.0101421132 chr3.0148710056 chr12.0113542149chr6.0100911125 chr17.0063553198 chr15.0040212792 chr12.0096253081chr12.0132963622 chr11.0002190999 chr11.0064014492 chr3.0065583873chr3.0147129333 chr22.0018846962 chr12.0007342661 chr8.0021645587chr3.0120004419 chr12.0058158604 chr15.0045428732 chr3.0089164229chr2.0091762598 chr11.0134525508 chr3.0072150129 chr7.0096642320chr10.0102589581 chr2.0066809303 chr12.0006446797 chr6.0166078201chr3.0050605386 chr6.0157390053 chr13.0026624812 chr11.0064757787chr2.0241151076 chr12.0051442996 chr14.0101963435 chr2.0172952158chr11.0012696865 chr9.0137354379 chr22.0049828000 chr14.0060975811chr12.0129282241 chr2.0080300303 chr20.0060100951 chr11.0073490719chr2.0157199292 chr12.0054338819 chr15.0093634307 chr12.0115122028chr15.0032829086 chr17.0021183474 chr11.0047600191 chr18.0035147994chr3.0019188810 chr19.0058520890 chr12.0000679392 chr22.0021922517chr7.0131229865 chr22.0050455644 chr6.0079793340 chr17.0019028901chr20.0034682243 chr14.0052746616 chr12.0054806459 chr5.0157965751chr22.0047784689 chr12.0057633739 chr9.0000973262 chr15.0020733814chr2.0157293096 chr17.0077775218 chr12.0054360615 chr6.0146350400chr9.0035972387 chr11.0126033819 chr9.0093864177 chr14.0021091166chr12.0093967107 chr5.0174402771 chr17.0045809660 chr21.0036258497chr18.0074827147 chr11.0118566515 chr11.0117865334 chr11.0031830385chr3.0048884826 chr17.0078944136 chr8.0086736710 chr2.0175616150chr18.0044556314 chr14.0105433783 chr13.0024828484 chr20.0050283865chr12.0066276259 chr18.0031803791 chr21.0045147607 chr11.0003185764chr11.0125011371 chr11.0107328843 chr3.0128565855 chr12.0056512395chr16.0057918264 chr12.0119616913 chr5.0172669936 chr7.0079764260chr14.0069262002 chr15.0059225979 chr5.0087988624 chr20.0055956988chr17.0059475373 chr15.0033023727 chr6.0003742885 chr16.0003152427chr18.0055106910 chr13.0031248541 chr15.0063570562 chr3.0038081164chr19.0034397576 chr21.0038083060 chr6.0050679720 chr11.0058975476chr6.0036410268 chr3.0185653023 chr11.0036237395 chr17.0001993730chr12.0000584675 chr12.0054391688 chr16.0032986491 chr18.0060028152chr9.0033082990 chr11.0089518999 chr2.0073518897 chr3.0119014121chr6.0073330966 chr10.0023479600 chr7.0137693710 chr16.0049891504chr16.0067876966 chr5.0153126277 chr3.0101232351 chr15.0096596722chr13.0027580970 chr15.0099104915 chr3.0018464995 chr11.0116114906chr6.0143832776 chr20.0060607587 chr11.0064948716 chr6.0075916565chr2.0214149450 chr17.0004047257 chr16.0087889984 chr8.0067025063chr8.0007114712 chr15.0024123323 chr3.0120461953 chr9.0019231893chr16.0029674073 chr14.0033401547 chr16.0014728089 chr16.0086884396chr17.0077216743 chr17.0046693239 chr20.0055532410 chr12.0006277039chr12.0014956526 chr14.0093132774 chr5.0134368249 chr11.0061463399chr3.0075690414 chr2.0090458454 chr22.0045018332 chr9.0120178198chr2.0149285701 chr16.0086538066 chr12.0058814470 chr6.0050804133chr7.0100465214 chr3.0068057166 chr12.0007071780 chr15.0078252068chr12.0069036581 chr17.0034754080 chr17.0035298681 chr13.0088327000chr14.0037428846 chr12.0130529772 chr11.0102702295 chr6.0036089235chr13.0113343518 chr17.0039464813 chr6.0118229764 chr18.0067957351chr12.0125005873 chr6.0038683255 chr17.0018759674 chr7.0104584531chr15.0090686530 The CpG list has been filtered for functional pathwayassignments. CpGs in hypothetical genes or unknown gene domains withunannotated functions were not included in this hard-copy listing, butall CpGs and their scores are retained in the database and are availablefor further analyses.

TABLE 4 Top 40 Hypermethylated Genes in Grpl. Genes are presented withPFAM and RefSeq name designations as well as KEGG pathway associations.MethyLoad UniProt HUGO Reactome 6843.2 C9JJ H3 unkUb-specific.processing.proteases 6050.5 Q8NGC1 OR11G2Olfactory.Signaling.Pathway 5274.3 Q8NGA5 OR10H4Olfactory.Signaling.Pathway 5238.1 Q8NH85 OR5R1Olfactory.Signaling.Pathway 5203.8 P10412 HIST1H1EFormation.of.Senescence-Associated.Heterochromatin.Foci. 4872.9 O95222OR6A2 Olfactory.Signaling.Pathway 4263.0 Q96RD0 OR8B2Olfactory.Signaling.Pathway 4263.0 Q8NHB1 OR2V1Olfactory.Signaling.Pathway 4125.6 Q8NGC3 OR10G2Olfactory.Signaling.Pathway 3767.1 Q8NGK5 OR52M1Olfactory.Signaling.Pathway 3059.6 Q8NGM9 OR8D4Olfactory.Signaling.Pathway 2705.2 Q8NGP3 OR5M9Olfactory.Signaling.Pathway 2613.2 P01563 IFNA2 TRAF6.mediated. IRF7.activation 2611.5 Q969M2 GJA10 Gap.junction.assembly 2552.5 P11021HSPA5 Antigen.Presentation:.Folding,.assembly.and.peptide.load 2481.8Q7L3B6 CDC37L1 Platelet.degranulation. 2356.3 Q9P032 NDUFAF4Complex.I.biogenesis 2339.5 Q8NGR5 OR1L4 Olfactory.Signaling.Pathway2261.1 Q15615 OR4D1 Olfactory.Signaling.Pathway 2251.5 Q8NGS6 OR13C3Olfactory.Signaling.Pathway 2203.3 Q8NH73 OR4S2Olfactory.Signaling.Pathway 2195.0 O95006 OR2F2Olfactory.Signaling.Pathway 2123.1 Q8NGY2 OR6K2Olfactory.Signaling.Pathway 2109.2 Q8NH18 OR5J2Olfactory.Signaling.Pathway 2107.0 Q53H54 TRMT12Synthesis.of.wybutosine.at.G37.of.tRNA(Phe) 2048.2 P14652 HOXB2Activation.of.anterior.HOX.genes.in.hindbrain.developmen 2032.4 Q15388TOMM20 Ub-specific.processing.proteases 2009.0 Q3LFD5 USP41ISG15.antivira.mechanism 1980.8 Q8NGD1 OR4N2 Olfactory.Signaling.Pathway1952.2 Q8NG41 NPB Peptide.ligand-binding.receptors 1936.0 Q8NGT7 OR2A12Olfactory.Signaling.Pathway 1918.2 Q5JQS5 OR2B11Olfactory.Signaling.Pathway 1856.2 P59535 TAS2R40G.alpha.(i).signaling.events 1794.2 Q8NGA6 OR10H5Olfactory.Signaling.Pathway 1780.2 P02533 KRT14Type.I.hemidesmosome.assembly 1777.8 Q8NGV0 OR2Y1Olfactory.Signaling.Pathway 1740.8 Q13145 BAMBIDownregulation.of.TFG-beta.receptor.signaling 1720.9 P01567 IFNA7TRAF6.mediated.IRF7.activation 1692.5 Q8NGX0 OR11L1Olfactory.Signaling.Pathway 1675.3 Q5H8A3 NMSG.alpha.(q).signaling.events The gene list has been filtered forfunctional pathway assignments. Hypothetical genes or genes withunannotated functions were not included in this hard-copy listing, butall genes and their scores are retained in the database and areavailable for further analyses.

TABLE 5 Top 40 Hypermethylated Genes in Grp2. Genes are presented withPFAM and RefSeq name designations as well as KEGG pathway associations.MethyLoad UniProt HUGO Reactome −7184.6 Q96R19 TAAR9G.alpha.(s).signalling.events −5915.6 Q8NGS3 OR1J1Olfactory.Signaling.Pathway −5203.1 O60404 OR10H3Olfactory.Signaling.Pathway −4655.8 Q8NGE9 OR9Q2Olfactory.Signaling.Pathway −4459.2 Q9H1M4 DEFB127 Defensins −4048.5Q9NV35 NU DT15 Phosphate.bond.hydrolysis.by.NUDT.proteins −3739.1 Q9NZP0OR6C3 Olfactory.Signaling.Pathway −3728.2 Q6IF42 OR2A2Olfactory.Signaling.Pathway −3590.9 Q96L21 RPL1OLNonsense.Mediated.Decay.(NMD).independent.of.the.Exon.Ju −3497.5 Q58HT5AWAT1 Wax.biosynthesis −3469.1 Q9H2C8 ORS1V1 Olfactory.Signaling.Pathway−3188.0 A6NL26 OR5B21 Olfactory.Signaling.Pathway −3047.1 Q8IXM3 MRPL41Mitochondrial.translation.initiation −2983.7 P09681 GIPG.alpha.(s).signalling.events −2873.1 Q6PGQ7 BORARegulation.of.PLK1.Activity.at.G2/M.Transition −2870.2 Q8NH60 OR52J3Olfactory.Signaling.Pathway −2831.0 Q96PE6 ZIM3Generic.Transcription.Pathway −2756.9 Q9HDD0 HRASLSAcyl.chain.remodelling.of.PE −2740.3 P12104 FABP2Hormone-sensitive.lipase.(HSL)-mediated.triacylglycerol. −2710.7 P00326ADH1C Ethanol.oxidation −2541.0 Q7Z7M8 B3GNT8O-linked.glycosylation.of.mucins −2535.7 A6NP11 ZNF716Generic.Transcription.Pathway −2444.0 Q8NGH7 OR52L1Olfactory.Signaling.Pathway −2435.9 Q6VVB1 NHLRC1Myoclonic.epilepsy.of.Lafora −2346.7 P82930 MRPS34Mitochondrial.translation.initiation −2324.1 O76100 OR7A10Olfactory.Signaling.Pathway −2194.5 Q8NG97 OR2Z1Olfactory.Signaling.Pathway −2181.1 Q8NGE5 OR10A7Olfactory.Signaling.Pathway −2157.6 Q6ZYL4 GTF2H5Dual.incision.in.TC-NER −2146.1 P62310 LSM3mRNA.Splicing.-.Major.Pathway −2017.1 Q8NG99 OR7G2Olfactory.Signaling.Pathway −2065.8 Q9NUP1 BLOC1S4Golgi.Associated.Vesicle.Biogenesis −2007.4 Q9BXT5 TEX15Meiotic.recombination −1971.8 Q6UXV4 APOOL Platelet.degranulation.−1963.6 P22680 CYP7A1Synthesis.of.bile.acids.and.bile.salts.via.27-hydroxycho −1933.4 P30939HTR1F G.alpha.(i).signaling.events −1909.8 P81277 PRLHPeptide.ligand-binding.receptors −1901.6 Q8NGS1 OR1J4Olfactory.Signaling.Pathway −1901.1 Q30KP8 DEFB136 Defensins −1798.3Q9NYY3 PLK2 TP53.regulates.transcription.of.additional.cell.cycle.ge Thegene list has been filtered for functional pathway assignments.Hypothetical genes or genes with unannotated functions were not includedin this hard-copy listing, but all genes and their scores are retainedin the database and are available for further analyses.

TABLE 6 Top 30 Hypermethylated Pathways in Grp1. Pathways are rankordered by methylation load score (ΔML) and include a list of the top 10scoring genes contributing to the pathways load value. The gene list isorganized in decreasing numerical order of the individual gene ΔMLscores MethyLoad Pathway Genes 1729.2 Trypotophan.metabolism unk 1518.0Rheumatoid.arthritis Q16552 1325.7 B.cell.receptor.signaling.path Q9UN191101.5 Streptomycin.biosynthesis 095455 1037.6Arrhythmogenic.right.ventricul P17302  869.7 Basal.cell.carcinoma P12643 794.6 Pantothenate.CoA.biosynthesis 095497  775.9Metabolism.xenobiotics.by.cyto 095154  745.9 Autoimmune.thyroid.diseaseA0A0R4J2F0  720.6 Chloroalkane.chloroalkene.degr P30837  679.7Neurotrophin.signaling.pathway unk  574.6 Aldosteroneregulated.sodium.reunk  543.8 Bisphenol.degradation unk  525.6Intestinal.immune.network.for P01833  509.1Amyotrophic.lateral.sclerosis. E7ESP9, P04040, P12036  498.8 PhagosomeQ5KU26  462.7 Tight.junction Q15334, P51153, P20337  442.6Fatty.acid.elongation Q9GZR5, Q9HB03  433.4 Reninangiotensin.system unk 418.1 Circadian.entrainment P48039, B7ZA25  413.5 Melanogenesis P14679,Q01726  406.7 Proximal.tubule.bicarbonate.re P49448  398.7Glutathione.metabolism Q96SL4  391.3 Vasopressinregulated.water.rea unk 370.0 Leishmaniasis P01583, P12314, P49006, P23458  356.6Collecting.duct.acid.secretion P51164, P02730  349.6Pancreatic.secretion P32238  348.5 Endocrine.other.factorregulate Unk 339.3 SNARE.interactions.in.vesicula O95183, O75558  339.1Phenylpropanoid.biosynthesis P30041 Gene order within each pathway isdetermined by decreasing methylation load scores ΔML). The first gene inthe list contributes the most to the pathway methylation score.

TABLE 7 Top 30 Hypermethylated Pathways in Grp2. Pathways are rankordered by methylation load score (ΔML) and include a list of the top 10scoring genes contributing to the pathways load value. The gene list isorganized in decreasing numerical order of the individual gene ΔMLscores. MethyLoad Pathway Genes −1238.0 Malaria P07996, Q12918 −1148.7Cell.cycle.yeast unk −1096.8 Twocomponent.system unk −1091.9Meiosis.yeast Q14566 −1036.6 Inositol.phosphate.metabolism Q8NFU5 −943.8 Taurine.hypotaurine.metabolism Q16878, Q96SZ5  −917.7Synaptic.vesicle.cycle Q7Z7G2  −898.0 Antigen.processing.presentatio unk   864.1 JakSTAT.signaling.pathway unk  −792.4 Phototransduction.fly unk −785.6 Valine.leucine.isoleucine.degr unk  −785.0 Atrazone.degradationunk  −740.7 Thyroid.cancer Unk  −676.8 Toluene.degradation Q96DG6 −669.8 Natural.killer.cell.mediated.c Q9BZM5, Q9BZM6  −666.2 Apoptosis014249  −653.2 Lipoic.acid.metabolism unk  −624.4 Base.excision.repairunk  −574.8 Nonhomologous.endjoining unk  −549.0Glycosphingolipid.biosynthesis 043505, P19526, Q9Y231, Q8NDV1  −539.4Glycosylphosphatidylinositolan unk  −492.8 Sulfur.relay.system Q7Z7A3,O95396  −481.5 Viral.carcinogenesis A0A0A0MSJ9, 014839, P51679  −461.6Fructose.mannose.metabolism Q9NQ88  −423.4 Mismatch.repair unk  −412.9Insulin.secretion P09681, P43220, Q8TDV5, 014842  −396.6Basal.transcription.factors P13984, Q15545, Q81ZX4  −392.3 PeroxisomeQ567V2, Q9BY49, P47989, P56589, P21549, 095822, Q9UBJ2, A8MXV4, Q15126,Q9UJM8  −382.9 Terpenoid.backbone.biosynthesi 075844, Q9BXS1  −363.1Methane.metabolism P05062 Gene order within each pathway is determinedby decreasing methylation load scores (ΔML). The first gene in the listcontributes the most to the pathway methylation score.

TABLE 8 Top 40 CpG Sites by P-value. Rank ordered listing of CpG sitesin known UniProt genes are sorted by P-values adjusted for falsediscovery rate (FDR) threshold. Site-specific dispersion was estimatedto equalize CpG variances. A Likelihood Ratio Test was used with adefined one-way ANOVA model for pairwise tests. FDR adj CpG Site IogFCP-val Response Gene Description chr9.0101559153 −1.26 2.38e−12   0.713ENSG00000165138 — chr2.0113240609 −2.18 2.19e−06 −0.42 TTL tubulintyrosine ligase chr12.0127210936   1.47 8.13e−05 −0.32 ENSG00000189238NP.001273148 chr11.0067173421   1.18 0.000   0.111 F5H037 —chr11.0065143966 −0.76 0.000   0.729 ENSG00000162241 NP.001265180chr6.0010695540   1.65 0.000 −0.42 PAK1IP1 PAK1 interacting protein 1chr3.0184302159   1.57 0.000 −0.58 H7C2X0 chr22.0039813308 −1.04 0.001  0.669 ENSG00000100324 NP.705717 chr3.0181428462 −1.89 0.001   0.556ENSG00000242808 NP.001177162 chr5.0141852077 −1.16 0.002   0.630ENSG00000231185 chr17.0014203257 −2.60 0.002 −0.82 ENSG00000125430NP.006032 chr18.0077960570   1.40 0.002 −0.30 K7ELH1 chr12.0031738230  1.02 0.002   0.638 ENSG00000170456 XP.005253385 chr3.0063987349 −0.680.002   0.775 ENSG00000163635 XP.005265422 chr10.0056713225   0.7610.002   0.693 E7EM53 chr16.0032675959 −1.52 0.004   0.581ENSG00000260311 XP.005255564 chr15.0089922792   1.37 0.004 −0.27ENSG00000255571 chr2.0000729785 −1.76 0.005   0.027 ENSG00000227713 —chr14.0087905699 −0.96 0.005   0.704 ENSG00000258859 — chr20.0000642737  1.18 0.005 −0.14 S4R3F8 — chr1.0094703313 −0.64 0.006   0.844ENSG00000137962 — chr2.0038724184 −0.94 0.006   0.652 ENSG00000231367 —chr8.0075917053   1.39 0.007   0.425 ENSG00000121005 NP.001273706chr14.0068286569 −2.95 0.007   0.299 A0A0A0MS18 chr17.0075087755 −1.260.007   0.102 ENSG00000234912 — chr15.0059785306   1.66 0.008   0.457A0A0U1RVI4 — chr6.0003738075 −0.77 0.008   0.665 PXDC1 PX domaincontaining 1 chr10.0104182130 −1.25 0.009   0.639 FBXL15 F-box andleucine rich repeat chr7.0097857306   0.614 0.013   0.718ENSG00000205356 XP.005250311 chr7.0150732834 −0.61 0.013   0.728ENSG00000197150 chr4.0057366782 −1.19 0.015   0.582 D6RDY6 —chr15.0082762246 −1.11 0.015   0.591 ENSG00000188384 NP.001157937chr16.0015680579 −0.66 0.015   0.704 ENSG00000166780 chr16.0002345736−0.56 0.018   0.761 ENSG00000167972 — chr19.0010597219 −0.64 0.019  0.717 K7EJ49 — chr3.0146250349 −0.69 0.019   0.727 ENSG00000188313 —chr12.0021958070   1.21 0.020   0.527 ENSG00000069431 — chr14.0023276073  1.07 0.020   0.526 G3V5A1 — chr22.0021922517   1.64 0.021   0.434ENSG00000185651 NP.001243285 chr15.0075401874   1.68 0.024 −0.71 H3BU63— logFC = log[2] of fold change; Response = up or down in Grp2 relativeto Grpl.Rankings of Sites for the Control Vs. DCIS Analysis

TABLE 9 Top 40 CpG Sites. Rank ordered listing of CpG sites contributingthe most to the ordinate discrimination in the NMDS analysis.  chr14.0068286569 chr11.0057407074 chr22.0042678985 chr5.0141488834chr18.0076481748 chr3.0181428462 chr15.0020733814 chr8.0086728497chr2.0000729785 chr22.0021922517 chr15.0034635088 chr15.0059785306chr20.0037899756 chr11.0093277585 chr17.0027912415 chr16.0032675959chr22.0037428734 chr12.0069036581 chr11.0027384364 chr8.0075917053chr2.0113240609 chr3.0057552599 chr16.0030366829 chr6.0163837639chr11.0064514192 chr11.0075111078 chr14.0073330009 chr2.0220665390chr17.0014666952 chr12.0067782441 chr12.0127210936 chr12.0057856280chr11.0079339302 chr6.0008613853 chr12.0053719703 chr13.0028056134chr20.0023474877 chr2.0204975304 chr7.0136556018 chr19.0016197423chr17.0072921703 chr11.0065774760 chr16.0033297106 chr14.0023057582chr5.0140782367 chr11.0063331532 chr8.0086556290 chr14.0019893202chr2.0110873784 chr18.0040499812 chr9.0035603644 chr15.0102480783chr9.0101559153 chr3.0184302159 chr16.0000766221 chr21.0015131488chr3.0032549768 chr2.0096810269 chr16.0001393584 chr4.0057366782chr2.0070315802 chr17.0080660575 chr6.0010695540 chr16.0012667415chr5.0001211402 chr11.0067173421 chr15.0065823539 chr17.0036286187chr12.0114476980 chr12.0021958070 chr6.0027794496 chr20.0004223373chr21.0038068930 chr16.0089544877 chr3.0038081164 chr13.0028550394chr3.0197382843 chr20.0040247763 chr11.0106894778 chr17.0050236261chr11.0134525508 chr16.0049891504 chr15.0023931943 chr2.0177053201chr16.0021512858 chr3.0039425054 chr3.0165780428 chr6.0080411829chr15.0085872107 chr14.0090798926 chr8.0001978959 chr14.0055903656chr17.0075087755 chr13.0114515221 chr2.0103775611 chrX.0118820440chr17.0036280378 chr5.0174027285 chr2.0095966379 chr16.0089831979chr17.0076136964 chr2.0074056574 chr19.0045458249 chr17.0014203257chr6.0026157100 chr12.0094361472 chr15.0047477411 chr2.0233370968chr12.0089918328 chr20.0031034124 chr11.0075989290 chr12.0065672210chr11.0067045324 chr16.0089516632 chr11.0009853771 chr6.0036089235chr5.0141852077 chr11.0009635545 chr22.0044280192 chr11.0134152806chr15.0059157852 chr13.0029647211 chr17.0043176656 chr3.0062365451chr18.0077960570 chr12.0006641855 chr16.0089554400 chr15.0082762246chr15.0090456975 chr2.0007837366 chr14.0080887882 chr5.0142574738chr2.0220384873 chr22.0019584506 chr22.0018371747 chr3.0180319542chr2.0065141315 chr14.0100005173 chr20.0047082324 chr18.0010122170chr22.0049764110 chr14.0037054111 chrX.0152973993 chr16.0005006250chr12.0119054118 chr11.0036237395 chr2.0006503608 chr9.0133309320chr18.0056589607 chr5.0077591726 chr5.0177002918 chr2.0073518897chr3.0039891004 chr18.0001303942 chr13.0053530320 chr14.0023276073chr6.0034192386 chr14.0061115424 chr8.0145743983 chr22.0021615439chr22.0048020255 chr11.0069323971 chr2.0220340677 chr20.0062778083chr11.0111460964 chr18.0035433091 chr16.0079802096 chr14.0024799383chr19.0004882313 chr20.0035243876 chr2.0202126440 chr20.0041844969chr12.0049958026 chr22.0044728672 chr13.0051569584 chr12.0075785138chr15.0089922792 chr15.0098961407 chr3.0011212205 chr6.0028864916chr2.0027775928 chr11.0049705897 chr6.0108486845 chr19.0004184748chr17.0012693967 chr12.0031738230 chr16.0089894315 chr3.0126214661chr19.0036037634 chr22.0039813308 chr11.0125249614 chr5.0151151794chr11.0001913079 chr15.0092935856 chr6.0160220177 chr11.0130701567chr13.0101091883 chr14.0020799205 chr11.0133835251 chr14.0097378133chr3.0182881438 chr14.0037410282 chr15.0042652379 chr6.0159572466chr12.0045271067 chr16.0000766588 chr6.0021597123 chr16.0086514423chr6.0168665883 chr15.0065702632 chr9.0037578967 chr3.0195837439chr3.0172166209 chr14.0074967635 chr3.0185619255 chr16.0089510331chr4.0190991714 chr6.0136481416 chr14.0102929042 chr20.0060296084chr17.0043663767 chr3.0108512761 chr17.0034491053 chr12.0032908207chr12.0030704058 chr9.0099540553 chr2.0216947053 chr10.0003514879chr18.0067634462 chr2.0086677764 chr14.0087905699 chr20.0058856822chr3.0095072859 chr17.0070270313 chr13.0111107585 chr2.0241905384chr14.0095008319 chr5.0141140612 chr15.0075495213 chr2.0175595489chr17.0030556864 chr13.0022709340 chr8.0121857592 chr3.0071354612chr15.0040212792 chr2.0000875249 chr18.0024586780 chr14.0084289630chr14.0058593798 chr20.0000642737 chr16.0082712158 chr11.0038893798chr5.0141048865 chr16.0011006670 chr20.0052164352 chr2.0229393549chr3.0013924461 chr20.0055721756 chr5.0126114228 chr12.0133186738chr22.0044729868 chr14.0099707257 chr12.0123424716 chr11.0044525297chr11.0045280326 chr11.0126226385 chr11.0129510558 chr6.0149324892chr3.0103361507 chr18.0068157667 chr22.0032044166 chr6.0079793340chr5.0157965751 chr20.0056431925 chr14.0070327521 chr12.0120827122chr2.0086609477 chr9.0102586890 chr22.0018834639 chr12.0014107327chr12.0066279288 chr13.0065785631 chr8.0015398239 chr2.0241640727chr12.0054379122 chr11.0118033212 chr7.0157964668 chr13.0048391192chr20.0036799918 chr13.0111766619 chr3.0082619041 chr16.0006822158chr5.0099954918 chr16.0011875269 chr20.0020837304 chr6.0163614535chr12.0042190273 chr15.0075401874 chr11.0103295615 chr19.0046993480chr12.0130155427 chr2.0042185584 chr18.0040051779 chr16.0089541352chr11.0061451702 chr13.0028549550 chr17.0016916346 chr16.0002205666chr18.0038227187 chr6.0031829093 chr2.0025018934 chr3.0181438281chr13.0066518415 chr6.0139349539 chr12.0067254364 chr3.0013899697chr22.0039627689 chr11.0119674018 chr14.0054457460 chr6.0035467808chr17.0026810215 chr15.0045423374 chr3.0061788180 chr11.0001949013chr22.0025490554 chr3.0075704892 chr3.0016536787 chr18.0024553398chr5.0025749975 chr14.0023652825 chr2.0124783828 chr16.0000774754chr11.0083236802 chr20.0033683440 chr16.0084043373 chr5.0061944346chr12.0115112086 chr17.0070370553 chr18.0029523502 chr2.0038724184chr20.0057075821 chr20.0031330621 chr22.0021612968 chr21.0037382085chr6.0000401429 chr12.0093494772 chr13.0088862433 chr18.0046477561chr20.0002827499 chr5.0141937225 chr13.0040770336 chr3.0171322486chr17.0080193825 chr5.0091190508 chr15.0045428732 chr17.0018996904chr18.0046303523 chr2.0118111021 chr5.0169624611 chr2.0058468651 The CpGlist has been filtered for functional pathway assignments. CpGs inhypothetical genes or unknown gene domains with unannotated functionswere not included in this hard-copy listing, but all CpGs and theirscores are retained in the database and are available for furtheranalyses.

TABLE 10 Top 40 Hypermethylated Genes in Grpl. Genes are presented withPFAM and RefSeq name designations as well as KEGG pathway associations.MethyLoad UniProt HUGO Reactome 3421.6 C9JJ H3 unkUb-specific.processing.proteases 3025.2 Q8NGC1 OR11G2Olfactory.Signaling.Pathway 2637.2 Q8NGA5 OR10H4Olfactory.Signaling.Pathway 2619.1 Q8NH85 OR5R1Olfactory.Signaling.Pathway 2601.9 P10412 HIST1H1EFormation.of.Senescence-Associated.Heterochromatin.Foci. 2436.4 095222OR6A2 Olfactory.Signaling.Pathway 2131.5 Q96RD0 OR8B2Olfactory.Signaling.Pathway 2131.5 Q8NHB1 OR2V1Olfactory.Signaling.Pathway 1883.5 Q8NGK5 OR52M1Olfactory.Signaling.Pathway 1529.8 Q8NGM9 OR8D4Olfactory.Signaling.Pathway 1352.6 Q8NGP3 OR5M9Olfactory.Signaling.Pathway 1306.6 P01563 IFNA2TRAF6.mediated.IRF7.activation 1305.8 Q969M2 GJA10 Gap.junction.assembly1276.3 P11021 HSPA5Antigen.Presentation:.Folding,.assembly.and.peptide.load 1240.9 Q7L3B6CDC37L1 Platelet.degranulation. 1169.8 Q8NGR5 OR1L4Olfactory.Signaling.Pathway 1130.5 Q15615 OR4D1Olfactory.Signaling.Pathway 1125.8 Q8NGS6 OR13C3Olfactory.Signaling.Pathway 1101.7 Q8NH73 OR4S2Olfactory.Signaling.Pathway 1097.5 095006 OR2F2Olfactory.Signaling.Pathway 1061.5 Q8NGY2 OR6K2Olfactory.Signaling.Pathway 1054.6 Q8NH18 OR5J2Olfactory.Signaling.Pathway 1004.5 Q3LFD5 USP41ISG15.antiviral.mechanism  990.4 Q8NGD1 OR4N2Olfactory.Signaling.Pathway  976.1 Q8NG41 NPBPeptide.ligand-binding.receptors  968.0 Q8NGT7 OR2Al2Olfactory.Signaling.Pathway  945.5 Q15617 OR8G1Olfactory.Signaling.Pathway  928.1 P59535 TAS2R40G.alpha.(i).signalling.events  897.1 Q8NGA6 OR1OH5Olfactory.Signaling.Pathway  890.1 P02533 KRT14 Type.1.hemidesmosome.assembly  888.9 Q8NGVO OR2Y1 Olfactory.Signaling.Pathway 870.4 Q13145 BAMBI Downregulation.of.TGF-beta.receptor.signaling  860.5P01567 IFNA7 TRAF6.mediated.IRF7.activation  846.2 Q8NGX0 OR11L1Olfactory.Signaling.Pathway  837.6 Q5H8A3 NMSG.alpha.(q).signalling.events  837.3 P14652 HOXB2Activation.of.anterior.HOX.genes.in.hindbrain.developmen  833.9 002539HIST1H1A Formation.of.Senescence-Associated.Heterochromatin.Foci.  800.7Q99626 CDX2 Synthesis,.secretion,.and.inactivation.of.Glucagon-like. 786.3 Q86XQ3 CATSPER3 Sperm.Motility.And.Taxes  785.4 Q969N4 TAAR8G.alpha.(s).signalling.events The gene list has been filtered forfunctional pathway assignments. Hypothetical genes or genes withunannotated functions were not included in this hard-copy listing, butall genes and their scores are retained in the database and areavailable for further analyses.

TABLE 11 Top 40 Hypermethylated Genes in Grp2. Genes are presented withPFAM and RefSeq name designations as well as KEGG pathway associations.MethyLoad UniProt HUGO Reactome −3592.3 Q96RI9 TAAR9G.alpha.(s).signalling.events −2957.8 Q8NGS3 OR1J1Olfactory.Signaling.Pathway −2601.6 060404 OR10H3Olfactory.Signaling.Pathway −2327.9 Q8NGE9 OR9Q2Olfactory.Signaling.Pathway −2279.6 Q9H1M4 DEFB127 Defensins −2024.2Q9NV35 NUDT15 Phosphate.bond.hydrolysis.by.NUDT.proteins −1869.5 Q9NZPOOR6C3 Olfactory.Signaling.Pathway −1864.1 Q6IF42 OR2A2Olfactory.Signaling.Pathway −1795.5 Q96L21 RPL1OLNonsense.Mediated.Decay.(NMD).independent.of.the.Exon.Ju −1748.8 Q58HT5AWAT1 Wax.biosynthesis −1734.5 Q9H2C8 OR51V1 Olfactory.Signaling.Pathway−1594.0 A6NL26 OR5B21 Olfactory.Signaling.Pathway −1523.5 Q8IXM3 MRPL41Mitochondrial.tmnslation.initiation −1491.8 P09681 GIPG.alpha.(s).signalling.events −1436.6 Q6PGQ7 BORARegulation.of.PLK1.Activity.at.G2/M.Transition −1435.1 Q8NH60 OR52J3Olfactory.Signaling.Pathway. −1415.5 Q96PE6 ZIM3Generic.Transcription.Pathway −1378.4 Q9H DDO HRASLSAcyl.chain.remodelling.of.PE −1370.2 P12104 FABP2Hormone-sensitive.lipase.(HSL)-mediated.triacylglycerol. −1355.3 P00326ADH1C Ethanol.oxidation −1271.6 P04118 CLPSRetinoid.metabolism.and.transport −1267.8 A6NP11 ZNF716Generic.Transcription.Pathway −1235.0 Q96G91 P2RY11G.alpha.(q).signalling.events −1222.0 Q8NGH7 OR52L1Olfactory.Signaling.Pathway −1218.0 Q6VVB1 NHLRC1Myoclonic.epilepsy.of.Lafora −1162.0 076100 OR7A10Olfactory.Signaling.Pathway −1090.5 Q8NGE5 OR10A7Olfactory.Signaling.Pathway −1078.8 Q6ZYL4 GTF2H5Dual.incision.in.TC-NER −1073.1 P62310 LSM3mRNA.Splicing.-.Major.Pathway −1053.5 Q8NG99 OR7G2Olfactory.Signaling.Pathway −1003.7 Q9BXT5 TEX15 Meiotic.recombination −985.9 Q6UXV4 APOOL Platelet.degranulation.  −981.8 P22680 CYP7A1Synthesis.of.bile.acids.and.bile.salts.via.27-hydroxycho  −966.7 P30939HTR1F G.alpha.(i).signalling.events  −954.9 P81277 PRLHPeptide.ligand-binding.receptors  −950.8 Q8NGS1 OR1J4Olfactory.Signaling.Pathway  −950.5 030KP8 DEFB136 Defensins  −899.2Q9NYY3 PLK2 TP53.regulates.transcription.of.additional.cell.cycle.ge −894.5 Q6ZNIO GCNT7 O-linked.glycosylation.of.mucins  −867.5 P61024CKS1B Cyclin.D.associated.events.in.G1 The gene list has been filteredfor functional pathway assignments. Hypothetical genes or genes withunannotated functions were not included in this hard-copy listing, butall genes and their scores are retained in the database and areavailable for further analyses.

TABLE 12 Top 30 Hypermethylated Pathways in Grp1. Pathways are rankordered by methylation load score (ΔML) and include a list of the top 10scoring genes contributing to the pathways load value. The gene list isorganized in decreasing numerical order of the individual gene ΔMLscores. MethyLoad Pathway Genes 759.0 Rheumatoid.arthritis Q16552 662.9B.cell.receptor.signaling.path Q9UN19 660.5 Tryptophan.metabolism unk550.7 Streptomycin.biosynthesis 095455 518.8Arrhythmogenic.right.ventricul P17302 410.6 Basal.cell.carcinoma P12643397.3 Pantothenate.CoA.biosynthesis 095497 372.9Autoimmune.thyroid.disease A0A0R4J2F0 363.9Metabolism.xenobiotics.by.cyto 095154 360.3Chloroalkane.chloroalkene.degr P30837 315.9Neurotrophin.signaling.pathway unk 287.3 Aldosteroneregulated.sodium.reunk 271.9 Bisphenol.degradation unk 268.9 Phagosome Q5KU26 267.1Proximal.tubule.bicarbonate.re P49448 262.8Intestinal.immune.network.for. P01833 234.6 Tight.junction Q15334,P51153, P20337 221.3 Fatty.acid.elongation Q9GZR5, Q9HB03 218.0Amyotrophic.lateral.sclerosis. E7ESP9, P04040, P12036 216.7Reninangiotensin.system unk 202.1 Circadian.entrainment P48039, B7ZA25199.4 Glutathione.metabolism Q96SL4 197.5 Vasopressinregulated.water.reaunk 194.8 Leishmaniasis P01583, P12314, P49006, P23458 189.4Collecting.duct.acid.secretion P51164, P02730 178.6 MelanogenesisP14679, 001726 175.3 Chemokine.signaling.pathway P10720, Q9Y4X3, O00626,P22362, O43927, P47992, P02776, P80075, 174.8 Pancreatic.secretionP32238 169.6 Phenylpropanoid.biosynthesis P30041 167.9 Novobiocinbiosynthesis P17735 Gene order within each pathway is determined bydecreasing methylation load scores (ΔML). The first gene in the listcontributes the most to the pathway methylation score.

TABLE 13 Top 30 Hypermethylated Pathways in Grp2. Pathways are rankordered by methylation load score (Δ M L) and include a list of the top10 scoring genes contributing to the pathways load value. The gene listis organized in decreasing numerical order of the individual gene Δ M Lscores. MethyLoad Pathway Genes −619.0 Malaria P07996, Q12918 −574.4Cell.cycle.yeast unk −546.0 Meiosis.yeast 014566 −518.3Inositol.phosphate.metabolism Q8NFU5 −470.1 Twocomponent.system unk−458.9 Synaptic.vesicle.cycle Q7Z7G2 −444.7Taurine.hypotaurine.metabolism 016878, Q96SZ5 −442.6Antigen.processing.presentatio unk −432.0 JakSTAT.signaling.pathway unk−392.5 Atrazine.degradation unk −370.3 Thyroid.cancer 016204 −353.1Nonhomologous.endjoining unk −350.9 Valine.leucine.isoleucine.degr unk−338.4 Toluene.degradation Q96DG6 −333.1 Apoptosis Q14249 −326.6Lipoic.acid.metabolism unk −291.4 Natural.killer.cell.mediated.c Q9BZM5,Q9BZM6 −289.4 Base.excision.repair unk −278.8 Phototransduction.fly unk−277.8 Glycosphingolipid.biosynthesis 043505, P19526, Q9Y231, Q8NDV1−269.7 Glycosylphosphatidylinositolan unk −214.7 Viral.carcinogenesisA0A0A0MSJ9, 014839, P51679 −212.2 African.trypanosomiasis Q6ZQW0,P55085, P22301 −204.2 Insulin.secretion P09681, P43220, Q8TDV5, 014842−196.2 Peroxisome Q567V2, Q9BY49, P47989, P56589, P21549, 095822,Q9UBJ2, A8MXV4, Q15126, Q9UJM8 −182.5 Mismatch.repair unk −182.1Sulfur.relay.system Q7Z7A3, 095396 −181.6 Methane.metabolism P05062−177.6 Terpenoid.backbone.biosynthesi 075844, Q9BXS1 −170.4 ShigellosisP60673 Gene order within each pathway is determined by decreasingmethylation load scores (Δ M L). The first gene in the list contributesthe most to the pathway methylation score.

TABLE 14 Top 40 CpG Sites by P-value. Rank ordered listing of CpG sitesin known UniProt genes are sorted by P-values adjusted for falsediscovery rate (FDR) threshold. Site-specific dispersion was estimatedto equalize CpG variances. A Likelihood Ratio Test was used with adefined one-way ANOVA model for pairwise tests. CpG Site logFC FDRadj-P-val Response Gene Description chr14.0068286569 −2.95 5.51e−20 downA0A0A0MS18 — chr11.0057407074 2.06 7.29e−13 up ENSG00000254602 —chr22.0042678985 −2.12 2.20e−11 down ENSG00000100207 — chr5.0141488834−1.96 1.19e−09 down NDFIP1 Nedd4 family interacting protechr3.0181428462 −1.89 4.54e−09 down ENSG00000242808 NP.001177162chr2.0000729785 −1.76 6.93e−08 down ENSG00000227713 — chr22.00219225171.64 8.49e−08 up ENSG00000185651 NP.001243285 chr15.0034635088 1.586.02e−07 up H0YM60 — chr15.0059785306 1.66 6.33e−07 up A0A0U1RVI4 —chr20.0037899756 1.58 6.43e−07 up ENSG00000211534 — chr17.0027912415−1.56 7.48e−06 down ENSG00000264031 NP.937790 chr16.0032675959 −1.529.69e−06 down ENSG00000260311 XP.005255564 chr12.0069036581 1.692.66e−05 up F5H7Y6 — chr11.0027384364 2.29 5.44e−05 up ENSG00000109881NP.542385 chr8.0075917053 1.39 7.34e−05 up ENSG00000121005 NP.001273706chr2.0113240609 −2.18 7.34e−05 down TTL tubulin tyrosine ligasechr16.0030366829 1.85 0.000 up ENSG00000260219 NP.001230575chr6.0163837639 1.73 0.000 up H0YGD6 — chr11.0064514192 1.34 0.000 upENSG00000068976 NP.005600 chr11.0075111078 2.19 0.000 up H0YF32 —chr14.0073330009 1.33 0.000 up ENSG00000205683 NP.036206chr12.0127210936 1.47 0.000 up ENSG00000189238 NP.001273148chr12.0057856280 −2.05 0.000 down ENSG00000111087 NP.001153517chr12.0053719703 2.22 0.000 up F8VV67 — chr20.0023474877 1.29 0.001 upCST8 cystatin 8 chr7.0136556018 1.25 0.001 up ENSG00000234352 —chr14.0023057582 −3.16 0.001 down F5GXX5 — chr19.0016197423 −1.39 0.001down ENSG00000167460 — chr17.0072921703 −1.43 0.001 down ENSG00000183034NP.835454 chr16.0033297106 −1.30 0.001 down ENSG00000262090 XP.005255564chr8.0086556290 −8.37 0.001 down ENSG00000270971 — chr5.0140782367 −1.940.001 down ENSG00000204956 NP.114382 chr14.0019893202 2.02 0.001 upENSG00000244306 NP.001005356 chr2.0110873784 −1.26 0.001 down F8WE57 —chr18.0040499812 −1.28 0.001 down ENSG00000152214 — chr9.0035603644 2.030.001 up ENSG00000107140 NP.006276 chr9.0101559153 −1.26 0.002 downENSG00000165138 — chr16.0000766221 1.45 0.002 up H3BUM1 —chr3.0184302159 1.57 0.002 up H7C2X0- chr3.0032549768 1.69 0.002 upENSG00000213849 NP.060271 logFC = log[2] of fold change; Response = upor down in Grp2 relative to Grp1.Rankings of Sites for the Control Vs. Invasive Breast Cancer Analysis

TABLE 15 Top 40 CpG Sites. Rank ordered listing of CpG sitescontributing the most to the ordinate discrimination in the NMDSanalysis. chr2.0113240609 chr11.0031846870 chr12.0050297405chr17.0073268308 chr9.0037578967 chr19.0042069923 chr3.0042911177chr4.0094751025 chr11.0007695679 chr14.0056407135 chr2.0086609477chr21.0045561163 chr14.0065689243 chr3.0033482718 chr15.0074630994chr17.0014203257 chr17.0042734551 chr10.0003514879 chr15.0035449759chr11.0017553236 chr11.0134341441 chr3.0105652482 chr14.0023057582chr2.0183731397 chr20.0000565588 chr12.0118518389 chr11.0043580678chr15.0057967982 chr13.0088862433 chr3.0071081288 chr2.0008833634chr11.0083251309 chr10.0056713225 chr13.0112720793 chr17.0077216743chr3.0070322314 chr20.0002584692 chr11.0125011371 chr15.0020733814chr3.0155945555 chr22.0042564578 chr19.0000925223 chr2.0101706746chr20.0049747671 chr15.0075362233 chr9.0127119764 chr15.0093634307chr3.0036074151 chr6.0085777767 chr11.0093707469 chr12.0074415833chr12.0005258607 chr6.0052456152 chr12.0002930852 chr20.0014745674chr14.0091072652 chr16.0085461847 chr13.0076387384 chr3.0028035684chr18.0008659584 chr5.0015758651 chr12.0050506409 chr18.0044774814chr20.0009496892 chr3.0138656356 chr16.0055539404 chr11.0068152468chr5.0003103410 chr13.0068862091 chr20.0046305398 chr3.0125872130chr17.0050236261 chr11.0036057669 chr11.0074125328 chr6.0073330966chr12.0113313505 chr19.0033752620 chr17.0017654366 chr8.0080740825chr6.0079793340 chr16.0088963738 chr16.0028935921 chr13.0093953330chr22.0049828000 chr5.0176889569 chr15.0043975270 chr2.0000729785chr15.0090638902 chr17.0021023063 chr8.0142485152 chr3.0132377906chr6.0079911924 chr3.0018465177 chr22.0023248614 chr5.0076928892chr5.0159604108 chr2.0073152645 chr14.0076953170 chr13.0113636156chr6.0038683255 chr12.0132671272 chr20.0055721756 chr2.0155639299chr16.0023763882 chr16.0027257120 chr17.0036280378 chr15.0066462172chr11.0075051113 chr11.0070512616 chr2.0220665390 chr15.0058173088chr2.0125572919 chr2.0020336517 chr13.0100075263 chr10.0116061880chr6.0021807995 chr14.0074967635 chr7.0102182417 chr11.0055979234chr22.0030973469 chr20.0042136963 chr20.0062486416 chr2.0071651284chr3.0000065214 chr20.0002827499 chr11.0009636058 chr7.0121048611chr16.0049888100 chr17.0007239160 chr5.0038246603 chr22.0044858743chr3.0054161016 chr13.0032377061 chr11.0127547963 chr20.0023017832chr3.0009113874 chr17.0007465780 chr2.0003743921 chr3.0064018099chr15.0101300081 chr17.0035447333 chr11.0044340428 chr12.0096632313chr3.0015674946 chr17.0078942509 chr18.0019747084 chr5.0121414025chr9.0129386447 chr22.0040316505 chr13.0022763192 chr19.0031744952chr12.0047401732 chr14.0101144066 chr13.0100648209 chr14.0061834869chr11.0111101254 chr11.0066816866 chr3.0126259929 chr9.0140657192chr20.0061554910 chr22.0018811858 chr17.0041322124 chr20.0010982737chr20.0057227608 chr20.0033683440 chr11.0134746717 chr7.0097857306chr6.0134210997 chr19.0045073719 chr12.0031738230 chr5.0007414317chr15.0042357486 chr2.0045029060 chr3.0134125941 chr4.0009215375chr20.0020742515 chr19.0039889239 chr19.0034397576 chr17.0079402697chr16.0065259721 chr2.0059252357 chr20.0044171320 chr12.0131694264chr17.0072174025 chr12.0013319824 chr20.0031261476 chr15.0058158361chr14.0035026525 chr2.0011767102 chr13.0082545364 chr5.0141309248chr11.0076370946 chr20.0061045252 chr14.0106416958 chr17.0066196740chr19.0041248943 chr12.0106631508 chr18.0076481748 chr2.0170276122chr3.0111632475 chr6.0003563820 chr18.0011284750 chr14.0070497701chr6.0112535805 chr8.0075917053 chr11.0001227691 chr5.0068950088chr11.0046564148 chr15.0075983228 chr18.0027861099 chr15.0048151460chr2.0068179208 chr12.0067143488 chr17.0062294803 chr12.0052961748chr11.0001945540 chr15.0069195045 chr16.0052458588 chr22.0032583409chr15.0071076473 chr22.0022296520 chr9.0116344785 chrX.0125349552chr14.0035099669 chr17.0045858885 chr13.0027455420 chr9.0090256959chr16.0086609680 chr20.0060909671 chr17.0016267297 chr14.0102929042chr11.0120384679 chr10.0060305797 chr9.0122756999 chr2.0016081660chr11.0134147815 chr11.0014384377 chr12.0089768127 chr11.0124669378chr14.0097592491 chr20.0062778083 chr12.0021958070 chr5.0148574900chr20.0055927974 chr1.0181104403 chr18.0052876612 chr11.0045202699chr12.0095162637 chr9.0100109657 chr16.0057337873 chr2.0029419721chr19.0011750942 chr20.0023032166 chr11.0044067218 chr13.0033591525chr7.0101939610 chr2.0236404318 chr11.0091598917 chr10.0119493401chr15.0059785306 chr14.0093527696 chr11.0009853771 chr2.0039665281chr22.0050455644 chr12.0072332759 chr12.0129282241 chr7.0098979512chr13.0022450119 chr17.0001954986 The CpG list has been filtered forfunctional pathway assignments. CpGs in hypothetical genes or unknowngene domains with unannotated functions were not included in thishard-copy listing, but all CpGs and their scores are retained in thedatabase and are available for further analyses.

TABLE 16 Top 40 Hypermethylated Genes in Grp1. Genes are presented withPFAM and RefSeq name designations as well as KEGG pathway associations.MethyLoad UniProt HUGO Reactome 3040.1 Q9NV35 NUDT15Phosphate.bond.hydrolysis.by.NUDT.proteins 2505.3 Q8NGC1 OR11G2Olfactory.Signaling.Pathway 2200.4 Q8NH73 OR4S2Olfactory.Signaling.Pathway 2104.4 C9JJH3 unkUb-specific.processing.proteases 2098.6 Q8NGD5 OR4K14Olfactory.Signaling.Pathway 2097.5 P10412 HIST1H1EFormation.of.Senescence-Associated.Heterochromatin.Foci. 2067.1 Q8NGR9OR1N2 Olfactory.Signaling.Pathway 1835.0 Q9P032 NDUFAF4Complex.I.biogenesis 1821.3 Q8NGP3 OR5M9 Olfactory.Signaling.Pathway1651.7 P08620 FGF4 FGFR3c.ligand.binding.and.activation 1592.7 Q8NH18OR5J2 Olfactory.Signaling.Pathway 1477.7 Q6IFN5 OR7E24Olfactory.Signaling.Pathway 1444.0 Q969N4 TAAR8G.alpha.(s).signalling.events 1298.9 Q96RD0 OR8B2Olfactory.Signaling.Pathway 1175.7 Q8N146 OR8H3Olfactory.Signaling.Pathway 1162.2 Q7L2Z9 CENPQDeposition.of.new.CENPA-containing.nucleosomes.at.the.ce 1130.3 O95222OR6A2 Olfactory.Signaling.Pathway 1095.9 Q9ULW2 FZD10Class.B/2.(Secretin.family.receptors) 1074.7 Q7RTS3 PTF1ARegulation.of.gene.expression.in.early.pancreatic.precur 1035.1 Q8NHB1OR2V1 Olfactory.Signaling.Pathway 1026.0 Q8NGA5 OR10H4Olfactory.Signaling.Pathway 1006.4 Q96RI8 TAAR6G.alpha.(s).signalling.events 993.9 Q8NGR5 OR1L4Olfactory.Signaling.Pathway 992.2 Q53H54 TRMT12Synthesis.of.wybutosine.at.G37.of.tRNA(Phe) 987.2 Q8NGE3 OR10P1Olfactory.Signaling.Pathway 984.1 Q9H208 OR10A2Olfactory.Signaling.Pathway 982.3 Q6P1L8 MRPL14Mitochondrial.translation.initiation 958.5 Q8NGJ4 OR52E2Olfactory.Signaling.Pathway 916.6 Q8NGH3 OR2D3Olfactory.Signaling.Pathway 910.4 P47985 UQCRFS1Respiratory.electron.transport 897.3 Q8NGW1 OR6B3Olfactory.Signaling.Pathway 886.0 Q8NGK4 OR52K1Olfactory.Signaling.Pathway 877.4 Q96RJ0 TAAR1G.alpha.(s).signalling.events 875.1 P52961 ART1 Alpha-defensins 867.3Q8NGS4 OR13F1 Olfactory.Signaling.Pathway 860.2 Q8NGY2 OR6K2Olfactory.Signaling.Pathway 852.4 Q8NGT7 OR2A12Olfactory.Signaling.Pathway 849.3 D6R901 unkUb-specific.processing.proteases 817.1 Q14973 SLC10A1Recycling.of.bile.acids.and.salts 813.0 Q9UBS3 DNAJB9XBP1(S).activates.chaperone.genes The gene list has been filtered forfunctional pathway assignments. Hypothetical genes or genes withunannotated functions were not included in this hard-copy listing, butall genes and their scores are retained in the database and areavailable for further analyses.

TABLE 17 Top 40 Hypermethylated Genes in Grp2. Genes are presented withPFAM and RefSeq name designations as well as KEGG pathway associations.MethyLoad UniProt HUGO Reactome −2613.2 Q8NGH7 OR52L1Olfactory.Signaling.Pathway −2458.3 Q8NGE9 OR9Q2Olfactory.Signaling.Pathway −2314.7 Q58HT5 AWAT1 Wax.biosynthesis−2272.4 Q8NH69 OR5W2 Olfactory.Signaling.Pathway −2132.1 Q8NGT0 OR13C9Olfactory.Signaling.Pathway −2014.5 Q96RI9 TAAR9G.alpha.(s).signalling.events −1869.9 Q96RA2 OR7D2Olfactory.Signaling.Pathway −1843.8 Q12918 KLRB1Immunoregulatory.interactions.between.a.Lymphoid.and.a.n −1809.7 Q30KQ1DEFB133 Defensins −1784.1 Q96PL1 SCGB3A2 Scavenging.by.Class.A.Receptors−1758.9 Q9Y2Y1 POLR3K RNA.Polymerase.III.Chain.Elongation −1664.5 P82930MRPS34 Mitochondrial.translation.initiation −1519.6 Q9HDD0 HRASLSAcyl.chain.remodelling.of.PE −1515.1 Q8IXM3 MRPL41Mitochondrial.translation.initiation −1355.5 O76100 OR7A10Olfactory.Signaling.Pathway −1342.8 Q9GZK3 OR2B2Olfactory.Signaling.Pathway −1331.8 Q6IF42 OR2A2Olfactory.Signaling.Pathway −1323.8 Q96L33 RHOV Rho.GTPase.cycle −1306.8Q30KQ7 DEFB113 Defensins −1267.0 Q6ZYL4 GTF2H5 Dual.incision.in.TC-NER−1263.4 Q8NGS3 OR1J1 Olfactory.Signaling.Pathway −1215.0 Q6IF63 OR52W1Olfactory.Signaling.Pathway −1190.3 Q8NGC3 OR10G2Olfactory.Signaling.Pathway −1178.5 Q96L21 RPL10LNonsense.Mediated.Decay.(NMD).independent.of.the.Exon.Ju −1163.4 Q8NGD4OR4K1 Olfactory.Signaling.Pathway −1155.0 Q8NG99 OR7G2Olfactory.Signaling.Pathway −1138.9 Q96A08 HIST1H2BARecruitment.and.ATM-mediated.phosphorylation.of.repair.a −1100.6 Q8NGQ4OR10Q1 Olfactory.Signaling.Pathway −1082.4 A6NHG9 OR5H14Olfactory.Signaling.Pathway −1074.7 Q9GZQ4 NMUR2G.alpha.(q).signalling.events −1074.0 Q9BYW3 DEFB126 Defensins −1004.4Q9NUP1 BLOC1S4 Golgi.Associated.Vesicle.Biogenesis −998.9 P62310 LSM3mRNA.Splicing.-.Major.Pathway −987.6 Q8NGP6 OR5M8Olfactory.Signaling.Pathway −974.2 Q96FJ2 DYNLL2Intraflagellar.transport −928.1 095221 OR5F1 Olfactory.Signaling.Pathway−926.7 Q8NGE5 OR10A7 Olfactory.Signaling.Pathway −914.4 Q8WZ84 OR8D1Olfactory.Signaling.Pathway −885.5 Q08ER8 ZNF543Generic.Transcription.Pathway −853.4 095813 CER1 Signaling.by.NODAL Thegene list has been filtered for functional pathway assignments.Hypothetical genes or genes with unannotated functions were not includedin this hard-copy listing, but all genes and their scores are retainedin the database and are available for further analyses.

TABLE 18 Top 30 Hypermethylated Pathways in Grp1. Pathways are rankordered by methylation load score (Δ M L) and include a list of the top10 scoring genes contributing to the pathways load value. The gene listis organized in decreasing numerical order of the individual gene Δ M Lscores. MethyLoad Pathway Genes 497.5 RNA.polymerase Q3B726 471.4Fructose.mannose.metabolism Q9NQ88 443.9 Aldosteroneregulated.sodium.reunk 433.9 Prion.diseases P11021, P10643 420.0 Tryptophan.metabolism unk372.4 Novobiocin.biosynthesis P17735 320.6 Prostate.cancer Q12778 320.1Chloroalkane.chloroalkene.degr P30837 319.0 Homologous.recombinationO43543 299.6 Leishmaniasis P49006, P01583, P23458, P12314 297.6Primary.bile.acid.biosynthesis O95992 291.2Neurotrophin.signaling.pathway unk 280.0 Streptomycin.biosynthesisO95455 258.2 ABC.transporters Q9NRK6 255.2 Circadian.rhythm O14503,P20393, Q9C0J9 244.9 Adipocytokine.signaling.pathwa P41159, Q86V24 236.5Biosynthesis.ansamycins Q9H0I9 234.6 Pantothenate.CoA.biosynthesisO95497 214.3 Lysine.degradation A0A0C4DFR3, A0A0A0MQV9 213.4Basal.cell.carcinoma P12643 213.3 Lipoic.acid.metabolism unk 211.7Carbohydrate.digestion.absorpt unk 210.4 Gap.junction Q9UKL4 208.8Arrhythmogenic.right.ventricul P17302 208.6Intestinal.immune.network.for. P01833 207.5 MAPK.signaling.pathwayQ05923, Q16690 204.8 Circadian.entrainment P48039, B7ZA25 198.0Progesteronemediated.oocyte.ma unk 174.5 Amino.sugar.nucleotide.sugar.mQ8TBE9 173.3 Glutathione.metabolism Q96SL4 Gene order within eachpathway is determined by decreasing methylation load scores. The firstgene in the list contributes the most to the pathway methylation score.

TABLE 19 Top 30 Hypermethylated Pathways in Grp2. Pathways are rankordered by methylation load score (Δ M L) and include a list of the top10 scoring genes contributing to the pathways load value. The gene listis organized in decreasing numerical order of the individual gene Δ M Lscores. MethyLoad Pathway Genes −764.8 Starch.sucrose.metabolism Q6PCE3−445.7 Butanoate.metabolism P0C7M7 −427.1 Antigen.processing.presentatiounk −423.1 Malaria QI2918, P07996 −418.1 Shigellosis P60673 −407.7Endocrine.other.factorregulate unk −399.6 Taurine.hypotaurine.metabolismQ16878, Q96SZ5 −367.2 Toluene.degradation Q96DG6 −366.8Glyeine.serine.threonine.metab unk −366.5 Valine.leucine.isoleucine.degrunk −328.9 Pancreatic.cancer unk −326.9 Oocyte.meiosis Q9UQE7 −317.4Glycosphingolipid.biosynthesis O43505, Q9Y231, P19526, Q8NDV1 −314.6Osteoclast.differentiation Q9HBY0 −288.8 Nonhomologous.endjoining unk−282.5 Sulfur.relay.system Q7Z7A3, O95396 −281.2Other.glycan.degradation Q9Y3R4, P04066 −250.2 Reninangiotensin.systemunk −240.5 Cell.cycle.yeast unk −226.2 Thyroid.cancer Q16204 −223.6Complement.coagulation.cascade P07204, P05160, P00748, P01008 −216.9Inositol.phosphate.metabolism Q8NFU5 −214.6 Small.cell.lung.cancerP33552, P61024 −198.3 Calcium.signaling.pathway unk −194.6Glycerolipid.metabolism Q17RR3, Q96AD5, O60218 −194.0African.trypanosomiasis Q6ZQW0, P55085, P22301 −193.8Natural.killer.cell.mediated.c Q9BZM5, Q9BZM6 −189.9 Protein.exportP61009 −185.2 Synaptic.vesicle.cycle Q7Z7G2 −183.8 Glutamatergic.synapseunk Gene order within each pathway is determined by decreasingmethylation load scores (Δ M L). The first gene in the list contributesthe most to the pathway methylation score.

TABLE 20 Top 40 CpG Sites by P-value. Rank ordered listing of CpG sitesin known UniProt genes are sorted by P-values adjusted for falsediscovery rate (FDR) threshold. Site-specific dispersion was estimatedto equalize CpG variances. A Likelihood Ratio Test was used with adefined one-way ANOVA model for pairwise tests. CpG Site logFC FDR adjpP-val Response Gene Description chr2.0113240609 −2.18 2.59e−11 down TTLtubulin tyrosine ligase chr12.0050297405 −1.30 2.27e−09 down F8VV65 —chr11.0031846870 −1.75 3.55e−09 down H0YDA4 — chr17.0073268308 −1.217.79e−07 down ENSG00000263843 — chr4.0094751025 0.991 4.85e−06 up ATOH1atonal bHLH transcription fact chr9.0037578967 1.49 4.97e−06 upENSG00000147912 — chr19.0042069923 −1.39 4.97e−06 down ENSG00000007129XP.005259429 chr11.0007695679 −1.35 4.97e−06 down ENSG00000166394 —chr21.0045561163 1.05 1.20e−05 up H7C2G3 — chr15.0074630994 0.9971.20e−05 up ENSG00000140459 — chr17.0042734551 −1.42 2.27e−05 downENSG00000180336 — chr17.0014203257 −2.23 5.20e−05 down ENSG00000125430NP.006032 chr12.0118518389 0.994 6.60e−05 up F5H724 — chr11.0134341441−1.33 6.89e−05 down ENSG00000255545 NP.061114 chr11.0017553236 −1.288.55e−05 down E9PNW1 — chr2.0183731397 1.83 0.000 up FRZBfrizzled-related protein chr3.0071081288 −1.05 0.000 downENSG00000114861 NP.001012523 chr10.0056713225 0.818 0.000 up E7EM53 —chr13.0088862433 1.23 0.000 up ENSG00000231019 — chr13.0112720793 −2.180.000 down SOX1 SRY-box 1 chr11.0043580678 −1.55 0.000 downENSG00000149084 — chr20.0002584692 1.18 0.000 up TMC2 transmembranechannel like 2 chr3.0155945555 −1.40 0.000 down H7C4S9 — chr3.0033482718−2.32 0.000 down C9JWL3 — chr20.0014745674 0.900 0.001 upENSG00000172264 — chr14.0091072652 1.09 0.001 up ENSG00000165914 —chr13.0076387384 0.828 0.001 up F8WD26 — chr12.0002930852 1.31 0.001 upENSG00000111203 XP.005253766 chr5.0015758651 0.966 0.001 up J3KNM9 —chr20.0046305398 0.777 0.001 up ENSG00000196562 XP.005260516chr12.0050506409 −1.57 0.001 down ENSG00000178449 XP.005269256chr20.0049747671 −1.00 0.001 down ENSG00000228820 — chr20.0009496892−1.49 0.002 down ENSG00000125869 NP.036393 chr6.0073330966 −1.09 0.002down A0A0A0MT07 — chr18.0044774814 2.04 0.002 up ENSG00000215474NP.001032891 chr3.0138656356 −1.87 0.002 down ENSG00000244578 —chr11.0036057669 1.18 0.002 up ENSG00000179241 NP.777562chr19.0000925223 −1.08 0.002 down K7EJ04 — chr2.0000729785 −1.45 0.003down ENSG00000227713 — chr17.0050236261 1.72 0.003 up ENSG00000154975 —logFC = log[2] of fold change; Response = up or down in Grp2 relative toGrp1.Rankings of Sites for the DCIS Vs. Invasive Analysis

TABLE 21 Top 40 CpG Sites. Rank ordered listing of CpG sitescontributing the most to the ordinate discrimination in the NMDSanalysis. CHR POS DOMAIN UniProt GO.terms chr3 120626543 upstr C9JQS3negative.regulation.; exocytosis; syntaxin.binding; regulate chr6139349539 upstr Q5SZC9 unk chr12 81330338 upstr H0YI92L27.domain.binding; inner.ear.developmen; exocytosis; synapt chr1161451702 intron F5GY58 endocannabinoid.sign; diacylglycerol.catab;neurotransmitter chr22 37680206 Na H0Y724 regulation.of.ARF.pr;ARF.guanyl-nucleotid; regulation.of.ce chr16 23653028 intron I3L0U8antigen.processing.a; chr17 38716331 intron J3KTN5 regulation.of.dendri;lymphocyte.migration; response.to.nitr chr14 38071393 intron Q3SY87protein.binding; chr3 184302159 intron H7C2X0 binding;positive.regulation.; translational.initia; astrocy chr6 10695540 intronQ9NWT1 negative.regulation.; protein.binding chr20 48730218 intronA0A0A0MSL3 regulation.of.DNA.re; nucleotide-binding.o; nucleotide-bindichr3 33482718 intron C9JWL3 viral.genome.replica; regulation.of.transc;angiogenesis; ne chr20 48553776 intron Q9Y508 spermatogenesis;metal.ion.binding; multicellular.organi; ce chr13 48612261 intron Q9NV358-oxo-7; nucleobase-containin; nucleobase-containin; GTP.cat chr1560297395 exon Q99853 cell.migration.in.di; mammary.gland.lobule;mammillothalamic chr11 64684954 upstr E9PQN5 binding;protein.phosphatase.; signal.transduction; activati chr18 77960570 exonK7ELH1 cell.junction.assemb; cell-cell.junction.o; tight.junction.achr11 41259310 intron E9PLP4 regulation.of.axonog; protein.binding chr1250616434 5utr F8VVQ7 zinc.ion.binding; actin.monomer.bindin;negative.regulation. chr3 155945555 exon H7C4S9 potassium.channel.re;synaptic.transmissio; voltage-gated.po chr11 130319714 exon Q8TE58zinc.ion.binding; proteolysis; metalloendopeptidase chr20 43513977 upstrQ4VY20 phosphoserine.bindin; negative.regulation.; cytoplasmic.sequchr14 29235148 5utr P55316 axon.midline.choice.; central.nervous.syst;forebrain.develo chr3 149689478 exon C9JQ45 regulation.of.synapt;negative.regulation.; positive.regulat chr12 55247863 intron Q96DR8post-translational.p; cellular.protein.met; O-glycan.process chr1131846870 intron H0YDA4 in.utero.embryonic.d; camera-type.eye.deve;calcium.ion.bind chr14 68286569 5utr A0A0A0MS18 DNA-dependent.ATPase;DNA.metabolic.proces; DNA.binding; ATP chr15 75401874 intron H3BU63phosphopantothenoyle; coenzyme.biosyntheti; pantothenate.met chr1829523502 exon J3QR00 ER.to.Golgi.vesicle-mediated.transport chr2230421733 intron Q8NFT8 glial.cell.different; Notch.receptor.proce;synapse.assembly chr20 58515736 intron A2A2M7 unk chr2 133426687 intronF8WD77 unk chr20 2821334 upstr X6RFT7 unk chr18 19284903 upstr Q86YT6heart.looping; positive.regulation.; blood.vessel.develop; n chr1296253081 intron F8W0W6 histone.mRNA.metabol; termination.of.RNA.p;ncRNA.metabolic. chr15 48625023 intron H0YMP1 dUTP.metabolic.proce;dUTP.diphosphatase.a; hydrolase.activi chr11 46938767 intron E9PNJ5synaptic.growth.at.n; receptor.tyrosine.ki; receptor.cluster chr1472980894 intron A0A0A0MRA9 positive.regulation.; regulation.of.G-prot;termination.of.G chr2 176947655 intron Q03828 limb.morphogenesis;sequence-specific.DN; regulation.of.tran chr14 78227825 intron F8WAD5unk The CpG list has been filtered for functional pathway assignments.CpGs in hypothetical genes or unknown gene domains with unannotatedfunctions were not included in this hard-copy listing, but all CpGs andtheir scores are retained in the database and are available for furtheranalyses.

TABLE 22 Top 40 Hypermethylated Genes in Grp1. Genes are presented withPFAM and RefSeq name designations as well as KEGG pathway associations.MethyLoad UniProt HUGO Reactome 5064.4 Q9NV35 NUDT15Phosphate.bond.hydrolysis.by.NUDT.proteins 2938.2 O60404 OR10H3Olfactory.Signaling.Pathway 2024.4 Q8N146 OR8H3Olfactory.Signaling.Pathway 1803.2 A6NL26 OR5B21Olfactory.Signaling.Pathway 1694.4 Q8NGS3 OR1J1Olfactory.Signaling.Pathway 1577.8 Q96RI9 TAAR9G.alpha.(s).signalling.events 1577.7 Q9H1M4 DEFB127 Defensins 1432.6Q8NGW1 OR6B3 Olfactory.Signaling.Pathway 1402.9 P12104 FABP2Hormone-sensitive.lipase.(HSL)-mediated.triacylglycerol. 1397.0 Q8NG97OR2Z1 Olfactory.Signaling.Pathway 1341.2 Q8NGE3 OR10P1Olfactory.Signaling.Pathway 1333.1 Q8NGR9 OR1N2Olfactory.Signaling.Pathway 1319.0 Q6ZNI0 GCNT7O-linked.glycosylation.of.mucins 1254.8 P61457 PCBD1Phenylalanine.and.tyrosine.catabolism 1227.2 P00326 ADH1CEthanol.oxidation 1213.1 P61024 CKS1B Cyclin.D.associated.events.in.G11206.8 Q6P1L8 MRPL14 Mitochondrial.translation.initiation 1189.8 Q8NH60OR52J3 Olfactory.Signaling.Pathway 1178.8 Q6UXV4 APOOLPlatelet.degranulation. 1174.1 P09681 GIP G.alpha.(s).signalling.events1163.4 Q9NZP0 OR6C3 Olfactory.Signaling.Pathway 1161.7 Q9NPF7 IL23ASignaling.by.Interleukins 1156.2 Q8NGG8 OR8B3Olfactory.Signaling.Pathway 1150.0 Q6PGQ7 BORARegulation.of.PLK1.Activity.at.G2/M.Transition 1132.0 Q96FX8 PERPTP53.regulates.transcription.of.several.additional.cell. 1098.8 Q8NH73OR4S2 Olfactory.Signaling.Pathway 1083.2 Q8NGX9 OR6P1Olfactory.Signaling.Pathway 1064.6 Q7RTS3 PTF1ARegulation.of.gene.expression.in.early.pancreatic.precur 1063.8 D6R901unk Ub-specific.processing.proteases 1054.5 Q8NGD5 OR4K14Olfactory.Signaling.Pathway 1045.9 Q9H2C8 OR51V1Olfactory.Signaling.Pathway 1041.8 Q9UHA7 IL36ASignaling.by.Interleukins 1033.9 Q7Z7M8 B3GNT8O-linked.glycosylation.of.mucins 1001.9 P08620 FGF4FGFR3c.ligand.binding.and.activation 977.2 Q9ULW2 FZD10Class.B/2.(Secretin.family.receptors) 973.6 Q8NGI7 OR10V1Olfactory.Signaling.Pathway 948.2 Q96PE6 ZIM3Generic.Transcription.Pathway 940.5 Q8N688 DEFB123 Defensins 911.9A6NP11 ZNF716 Generic.Transcription.Pathway 902.5 Q8NGH3 OR2D3Olfactory.Signaling.Pathway The gene list has been filtered forfunctional pathway assignments. Hypothetical genes or genes withunannotated functions were not included in this hard-copy listing, butall genes and their scores are retained in the database and areavailable for further analyses.

TABLE 23 Top 40 Hypermethylated Genes in Grp2. Genes are presented withPFAM and RefSeq name designations as well as KEGG pathway associations.MethyLoad UniProt HUGO Reactome −3253.1 Q8NGC3 OR10G2Olfactory.Signaling.Pathway −3197.0 Q8NH85 OR5R1Olfactory.Signaling.Pathway −2289.2 Q30KQ1 DEFB133 Defensins −2275.2Q8NGK5 OR52M1 Olfactory.Signaling.Pathway −2108.9 Q8NGT0 OR13C9Olfactory.Signaling.Pathway −1778.5 Q8NGD4 OR4K1Olfactory.Signaling.Pathway −1744.1 Q8NH69 OR5W2Olfactory.Signaling.Pathway −1698.9 Q12918 KLRB1Immunoregulatory.interactions.between.a.Lymphoid.and.a.n −1672.6 Q969M2GJA10 Gap.junction.assembly −1625.6 O95221 OR5F1Olfactory.Signaling.Pathway −1611.2 Q8NGA5 OR10H4Olfactory.Signaling.Pathway −1391.2 Q8NGH7 OR52L1Olfactory.Signaling.Pathway −1359.7 Q8NG94 OR11H1Olfactory.Signaling.Pathway −1317.2 C9JJH3 unkUb-specific.processing.proteases −1306.1 O95222 OR6A2Olfactory.Signaling. Pathway −1213.5 Q86XQ3 CATSPER3Sperm.Motility.And.Taxes −1211.3 Q9NYW4 TAS2R5G.alpha.(i).signalling.events −1192.3 Q969Q0 RPL36ALNonsense.Mediated.Decay.(NMD).independent.of.the.Exon.Ju −1189.8 Q5TF39MFSD4B Na+-dependent.glucose.transporters −1183.1 Q9GZK3 OR2B2Olfactory.Signaling.Pathway −1176.3 Q9BYW3 DEFB126 Defensins −1175.2Q96PL1 SCGB3A2 Scavenging.by.Class.A.Receptors −1096.4 Q8NHB1 OR2V1Olfactory.Signaling.Pathway −1092.4 Q96RA2 OR7D2Olfactory.Signaling.Pathway −1082.1 Q30KQ7 DEFB113 Defensins −1080.4Q8NGD1 OR4N2 Olfactory.Signaling.Pathway −1075.4 Q9Y6L6 SLCO1B1Defective.SLCO1B1.causes.hyperbilimbinemia,.Rotor.type. −1070.2 Q96FJ2DYNLL2 Intraflagellar.transport −1046.3 Q9Y2Y1 POLR3KRNA.Polymerase.III.Chain.Elongation −1045.2 Q8NGM9 OR8D4Olfactory.Signaling.Pathway −1023.9 Q8NGQ4 OR10Q1Olfactory.Signaling.Pathway −982.8 P14652 HOXB2Activation.of.anterior.HOX.genes.in.hindbrain.developmen −969.4 Q8NGP6OR5M8 Olfactory.Signaling.Pathway −957.6 Q9UQK1 PPP1R3CMyoclonic.epilepsy.of.Lafora −951.1 P01563 IFNA2TRAF6.mediated.IRF7.activation −919.7 Q7L3B6 CDC37L1Platelet.degranulation. −912.2 Q9H082 RAB33B Intra-Golgi.traffic −900.2A6NL08 OR6C75 Olfactory.Signaling.Pathway −888.4 P61952 GNG11G.alpha.(q).signalling.events −875.8 Q16552 IL17AInterleukin-17.signaling The gene list has been filtered for functionalpathway assignments. Hypothetical genes or genes with unannotatedfunctions were not included in this hard-copy listing, but all genes andtheir scores are retained in the database and are available for furtheranalyses.

TABLE 24 Top 30 Hypermethylated Pathways in Grp1. Pathways are rankordered by methylation load score (ΔML) and include a list of the top 10scoring genes contributing to the pathways load value. The gene list isorganized in decreasing numerical order of the individual gene ΔMLscores. MethyLoad Pathway Genes 830.3 Fructose.mannose.metabolism Q9NQ88704.7 Lipoic.acid.metabolism Unk 586.9 Meiosis.yeast Q14566 518.1Twocomponent.system Unk 441.8 Base.excision.repair unk 406.8Biosynthesis.ansamycins Q9H0I9 394.1 Atrazine.degradation unk 386.1Apoptosis Q14249 381.7 Phototransduction.fly unk 333.9 Cell.cycle.yeastunk 324.1 Inositol.phosphate.metabolism Q8NFU5 308.5Primary.bile.acid.biosynthesis O95992 307.2 JakSTAT.signaling.pathwayunk 298.4 Glycerophospholipid.metabolism Q9Y259 274.0Amino.sugar.nucleotide.sugar.m Q8TBE9 260.2 Synaptic.vesicle.cycleQ7Z7G2 239.4 Prostate.cancer Q12778 236.6 Prion.diseases P10643, P11021232.8 Pyrimidine.metabolism P56597, Q02127 230.3Nucleotide.excision.repair R4GMW8, P41208, Q6ZYL4 229.5mTOR.signaling.pathway Q7L523, Q9NX09 224.0 Mismatch.repair unk 213.2Glycosylphosphatidylinositolan unk 211.9 Terpenoid.backbone.biosynthesiO75844, Q9BXS1 204.5 Novobiocin.biosynthesis P17735 203.1Viral.carcinogenesis Q14839, A0A0A0MSJ9, P51679 198.5Selenocompound.metabolism O43252 192.2 Malaria P07996, Q12918 180.1Sphingolipid.metabolism Q8TDN7, Q16739, Q9BX95, Q8IWX5 179.2Pathogenic.Escherichia.coli.in Q9H4B7, P68371, Q9BVA1, Q6PEY2, Q13885Gene order within each pathway is determined by decreasing methylationload scores (_ML). The first gene in the list contributes the most tothe pathway methylation score.

TABLE 25 Top 30 Hypermethylated Pathways in Grp2. Pathways are rankordered by methylation load score (ΔML) and include a list of the top 10scoring genes contributing to the pathways load value. The gene list isorganized in decreasing numerical order of the individual gene ΔMLscores. MethyLoad Pathway Genes −875.8 Rheumatoid.arthritis Q16552−748.0 Starch.sucrose.metabolism Q6PCE3 −490.8B.cell.receptor.signaling.path Q9UN19 −466.9 Reninangiotensin.system unk−444.6 Tryptophan.metabolism unk −362.4 Other.glycan.degradation Q9Y3R4,P04066 −345.9 Endocrine.other.factorregulate unk −342.1Autoimmune.thyroid.disease A0A0R4J2F0 −339.8 Bisphenol.degradation Unk−338.0 Oocyte.meiosis Q9UQE7 −320.4 Melanogenesis P14679, Q01726 −309.9Arrhythmogenic.right.ventricul P17302 −308.2 Phagosome Q5KU26 −290.1Osteoclast.differentiation Q9HBY0 −277.7 Butanoate.metabolism P0C7M7−276.7 Complement.coagulation.cascade P07204, P00748, P01008, P05160−270.7 Streptomycin.biosynthesis O95455 −267.2 RNA.polymerase Q3B726−263.6 Metabolism.xenobiotics.by.cyto O95154 −256.1 Pancreatic.cancerunk −247.7 Shigellosis P60673 −204.8 Basal.cell.carcinoma P12643 −203.9Hepatitis.B P14780, Q13233, O43889, P60484 −201.8 Small.cell.lung.cancerP33552, P61024 −197.8 Chemokine.signaling.pathway O00626, P10720,Q9Y4X3, O43927, P80075, P22362, P47992, P02776 −197.8Glycine.serine.threonine.metab unk −192.9 Folate.biosynthesis P35270,Q92820 −192.6 Hematopoietic.cell.lineage P06126, P07359, P01588, P15813,P29016, P14770 −186.1 Pantothenate.CoA.biosynthesis O95497 −184.6Gastric.acid.secretion Q6AI14, P78508, Q9Y6J6, P61278 Gene order withineach pathway is determined by decreasing methylation load scores (_ML).The first gene in the list contributes the most to the pathwaymethylation score.

TABLE 26 Top 40 CpG Sites by P-value. Rank ordered listing of CpG sitesin known UniProt genes are sorted by P-values adjusted for falsediscovery rate (FDR) threshold. Site-specific dispersion was estimatedto equalize CpG variances. A Likelihood Ratio Test was used with adefined one-way ANOVA model for pairwise tests. CpG Site logFC FDR adjP-val Response Gene Description chr20.0062282831 −1.77 4.24e−19 downENSG00000197457 NP.001263239 chr3.0119422009 1.90 1.71e−16 up H7C5C8 —chr10.0104182130 1.46 4.72e−10 up FBXL15 F-box and leucine rich repeatchr16.0031227255 −2.11 5.42e−10 down ENSG00000177238 NP.001008275chr9.0101559153 1.27 2.19e−08 up ENSG00000165138 — chr12.0002862275−2.02 4.79e−08 down ENSG00000256150 — chr16.0029460831 1.15 8.03e−08 upENSG00000198106 — chr11.0118306730 −1.55 1.07e−07 down ENSG00000118058 —chr12.0113573398 −1.74 1.70e−07 down ENSG00000111344 — chr14.0069261542−2.31 3.94e−07 down ENSG00000185650 — chr15.0101300081 −1.66 7.51e−07down ENSG00000259579 NP.078984 chr3.0184302159 −1.67 1.01e−06 downH7C2X0 — chr20.0041428080 1.10 1.26e−06 up B1AJS0 — chr12.00058858131.05 1.84e−06 up F5GXT3 — chr2.0177014555 −2.09 2.66e−06 downENSG00000128652 NP.008829 chr13.0048612261 −1.57 2.71e−06 down NUDT15nudix hydrolase 15 chr12.0127210936 −1.40 6.91e−06 down ENSG00000189238NP.001273148 chr6.0010695540 −1.61 7.28e−06 down PAK1IP1 PAK1interacting protein 1 chr3.0065583873 1.57 8.97e−06 up ENSG00000151276 —chr20.0004155798 0.990 9.09e−06 up Q5TE25 — chr18.0077960570 −1.509.32e−06 down K7ELH1 — chr11.0060971100 1.20 1.18e−05 up ENSG00000229859— chr11.0067173421 −1.17 2.82e−05 down F5H037 — chr22.0037680206 −1.904.01e−05 down H0Y724 — chr2.0225266367 2.00 9.37e−05 up ENSG00000124019— chr3.0107308392 1.80 9.37e−05 up H7C1Q0 — chr22.0028542468 1.059.37e−05 up B0QYP4 — chr2.0189618980 0.963 9.55e−05 up ENSG00000223523 —chr20.0031592073 0.850 9.55e−05 up Q5TDX8 — chr16.0001877823 −1.53 0.000down ENSG00000180185 NP.112485 chr12.0050279079 −1.13 0.000 down F8VV65— chr1.0015251350 0.672 0.000 up ENSG00000189337 NP.001018001chr2.0242004042 1.39 0.000 up B5MEF5 — chr6.0126661739 −2.22 0.000 downENSG00000203760 — chr17.0080708405 −1.09 0.000 down FN3K fructosamine 3kinase chr3.0120626543 −2.11 0.000 down C9JQS3 — chr16.0056671862 1.400.000 up ENSG00000205362 NP.005937 chr19.0020387066 1.28 0.000 upENSG00000267383 NP.009069 chr21.0045721062 −1.46 0.000 downENSG00000141959 — chr3.0124976593 1.28 0.000 up ENSG00000221955 — logFC= log[2] of fold change; Response = up or down in Grp2 relative to Grp1.

TABLE 27 Top 1000 CpG sites used used for prediction of risk ofinvasiveness in blind study and ranked by p-value. CHRPOS logFC PValuechr20.0005100198 1.42179995 2.24E−13 chr3.0116170128 −1.1460506 5.32E−13chr2.0136583332 0.89282221 5.50E−12 chr18.0003771925 −2.0448811 1.39E−10chr14.0024615469 −2.2607198 8.23E−10 chr19.0005075863 −1.12441658.82E−10 chr5.0056352628 0.84421582 2.32E−09 chr7.0131268894 −1.09054343.02E−09 chrX.0047343019 0.74231529 4.86E−09 chr3.0139086222 −0.77298749.29E−09 chr9.0111238265 −0.7120328 1.51E−08 chr7.0028605794 −1.09728121.81E−08 chr12.0133443239 −1.1540247 2.19E−08 chr13.00795511730.82890047 2.38E−08 chr22.0046305438 0.89464641 2.54E−08chr15.0097151927 1.1072284 3.53E−08 chr11.0048200842 0.73716749 4.48E−08chr2.0009898288 0.99226365 4.50E−08 chr2.0121118677 0.63220566 4.70E−08chr17.0070760064 0.65880467 5.57E−08 chr20.0007041339 0.962586226.08E−08 chr2.0229878578 0.70667337 6.17E−08 chr6.0167651348 −1.57121327.21E−08 chr3.0159375789 0.80887734 7.61E−08 chr16.0021610035 2.40503927.85E−08 chr13.0058888451 0.7715333 8.75E−08 chr22.0041761752 0.914498148.83E−08 chr16.0074460552 0.9557071 1.01E−07 chr8.0000224441 −0.99396281.04E−07 chr11.0045197654 0.73057185 1.50E−07 chr7.0004020430 0.732959981.55E−07 chr11.0044338492 −1.1433836 1.66E−07 chr6.0008173236 0.703714491.86E−07 chr17.0006605234 −1.06738 2.21E−07 chr6.0127166748 0.710743023.33E−07 chr5.0107735329 0.77293713 3.35E−07 chr12.0098725299 0.948721663.63E−07 chrX.0145243680 0.55365716 3.97E−07 chr14.0103403232 0.669246574.58E−07 chr16.0004816546 1.2271846 4.59E−07 chr5.0000082900 −0.73965164.65E−07 chr2.0048943971 0.57157878 4.66E−07 chr12.0081923492 0.78616115.47E−07 chr19.0055836171 −0.7943521 5.60E−07 chr17.00778358720.87262222 6.81E−07 chr1.0156416942 0.49841039 7.47E−07 chr3.01837718360.70394904 7.58E−07 chr5.0004939235 −0.7432739 8.15E−07 chr5.0137610361−1.0255363 8.49E−07 chr2.0237602634 −0.7307519 8.50E−07 chr9.0137961816−0.7393457 8.64E−07 chr16.0075145905 1.418913 9.57E−07 chr18.00556276130.72690684 9.78E−07 chr6.0164722394 0.73010958 1.00E−06 chr20.00362990830.7096724 1.01E−06 chr11.0122900154 0.69933001 1.03E−06 chr6.00462952190.79927637 1.08E−06 chr16.0058529353 −1.2677448 1.09E−06chr12.0122230944 −1.2872705 1.14E−06 chr5.0142142127 −0.8478472 1.14E−06chr3.0061703710 −0.6220134 1.15E−06 chr14.0094286835 0.71267469 1.17E−06chr19.0007936720 1.29180551 1.31E−06 chr20.0037591461 −1.62788071.39E−06 chr11.0062767705 −0.7265815 1.44E−06 chr2.0233308839 0.662556651.54E−06 chr4.0045155506 0.54310837 1.57E−06 chr9.0000159680 −1.25153951.63E−06 chr13.0048449705 −0.7572854 1.78E−06 chr20.0059026704−1.1675921 1.84E−06 chr15.0059817268 −0.8535793 1.89E−06chr13.0087178246 0.89266193 1.89E−06 chr3.0126257511 −0.6324197 1.96E−06chr14.0102783036 −1.7689019 1.96E−06 chr2.0027579337 −1.1682393 1.97E−06chr7.0116273352 0.81598333 2.00E−06 chr2.0128663833 −0.6209 2.07E−06chr5.0100843674 0.68437251 2.07E−06 chr11.0119724615 0.69053413 2.08E−06chr3.0045133805 0.66174842 2.22E−06 chr16.0076936044 0.7279669 2.29E−06chr5.0140870213 −0.6006419 2.30E−06 chr5.0159804189 0.7318523 2.32E−06chr11.0125754851 0.71063912 2.40E−06 chr11.0115624765 0.779060242.44E−06 chr18.0033675411 0.74760036 2.49E−06 chr16.00298680440.66920749 2.57E−06 chr9.0012612775 0.73600706 2.68E−06 chr18.00470579310.67571035 2.80E−06 chr7.0042432323 0.67178647 2.85E−06 chr5.00992200170.87458057 2.96E−06 chr11.0077185588 1.19357809 3.00E−06 chr7.0130133579−1.0839047 3.04E−06 chr8.0144976814 −1.059382 3.16E−06 chr11.00652388700.70757985 3.16E−06 chr18.0037258709 0.85653168 3.27E−06chr17.0002439051 −0.7574154 3.39E−06 chr9.0115848647 −1.5003321 3.41E−06chr6.0004647548 −0.913113 3.41E−06 chr11.0038245360 0.7180336 3.41E−06chr10.0132675670 0.64545599 3.41E−06 chr2.0218661279 0.64103416 3.45E−06chr2.0036165222 0.77704591 3.58E−06 chr5.0053096496 0.67029349 3.60E−06chr3.0131485356 0.85503524 3.72E−06 chr20.0051202616 0.82505759 3.78E−06chr8.0061326266 −0.8922994 3.79E−06 chr22.0026176223 0.68985137 3.79E−06chr20.0048452918 −0.7392151 3.87E−06 chr17.0031989964 −0.88472643.93E−06 chr10.0034801856 0.61469321 4.27E−06 chr8.0032312701 0.720322874.41E−06 chr11.0026638613 0.63297 4.42E−06 chr15.0079827069 0.788955544.47E−06 chr18.0010781190 0.84285991 4.48E−06 chr13.00897436820.67122942 4.52E−06 chr10.0089168004 0.93459378 4.53E−06chr13.0045652971 −0.9397673 4.76E−06 chr20.0045811892 −0.77386184.82E−06 chr5.0090438852 0.68125096 4.83E−06 chr16.0065543654 0.767012614.85E−06 chr12.0070329407 0.71880582 4.86E−06 chr15.0096206901 0.77550524.87E−06 chr14.0060388658 −0.6959306 4.94E−06 chr20.0061030211−1.5240984 5.02E−06 chr18.0022686352 0.88560794 5.19E−06chr15.0041075917 0.73011358 5.24E−06 chr14.0024641797 0.8965562 5.27E−06chr3.0100054571 −0.7327839 5.39E−06 chr5.0084824603 0.76840845 5.53E−06chr6.0167335873 0.70793696 5.53E−06 chr10.0099283204 0.57109819 5.61E−06chr2.0177258508 0.60913373 5.72E−06 chr12.0062860430 1.20186348 5.80E−06chr5.0172785376 0.72865225 5.83E−06 chr5.0157435312 0.72818783 5.95E−06chr18.0040240768 0.67018018 5.99E−06 chr12.0123194517 0.639600156.00E−06 chr7.0026838131 0.72219973 6.01E−06 chr6.0032096599 0.944966266.05E−06 chr9.0139561883 −0.686308 6.29E−06 chr3.0090052788 0.683086336.31E−06 chr10.0124234752 −0.7283645 6.36E−06 chr19.00122719700.61821339 6.47E−06 chr10.0132926074 −0.8299413 6.53E−06chr10.0087833424 0.66297996 6.80E−06 chr18.0019760779 −0.95423266.82E−06 chr20.0026221421 0.74389756 6.96E−06 chr5.0113816235 0.75869966.98E−06 chr15.0053965908 0.64101162 6.99E−06 chr19.0010767724−0.8440964 7.25E−06 chr3.0023652434 0.61929288 7.52E−06 chr11.00351104870.70515095 7.59E−06 chr22.0037200902 0.70733823 7.75E−06chr11.0030394645 0.61008756 7.77E−06 chr18.0037578424 −0.84207577.90E−06 chr15.0026276337 0.64924394 8.06E−06 chr19.00508775560.60873423 8.09E−06 chr3.0045349563 0.61674908 8.15E−06 chr3.01792709600.58476677 8.17E−06 chr12.0113853884 0.66782419 8.41E−06chr15.0039286447 0.79591802 8.52E−06 chr17.0058090391 −0.58477328.73E−06 chr1.0154910059 −0.5000733 8.82E−06 chr20.0019006992 0.784156899.20E−06 chr5.0003741039 −0.6492828 9.71E−06 chrX.0095583828 0.514628119.74E−06 chr3.0031900140 0.97626505 9.82E−06 chr7.0150544980 0.628181959.92E−06 chr9.0133887499 −1.0632639 9.94E−06 chr14.0024713193 0.767248731.00E−05 chr3.0013836751 0.700824 1.02E−05 chr6.0015480087 0.685301611.03E−05 chr2.0117820185 0.5477872 1.05E−05 chr12.0072332759 −1.12274371.06E−05 chr2.0010092513 −0.6775732 1.08E−05 chr16.0004948620 0.764101361.14E−05 chr18.0010344859 0.65708802 1.14E−05 chr14.00619098460.65127534 1.15E−05 chr5.0167798272 0.72353008 1.18E−05 chr2.01294521640.63085607 1.20E−05 chr19.0032122322 −0.6052487 1.20E−05chr10.0135055821 0.59828613 1.21E−05 chr13.0032990677 −0.66048291.21E−05 chr18.0000318921 0.6420835 1.23E−05 chr17.0056584557 0.655173541.24E−05 chr18.0044554788 1.72193398 1.26E−05 chr11.00788702980.70383423 1.28E−05 chr5.0095160390 −1.423951 1.29E−05 chr3.01976767762.0746083 1.32E−05 chr18.0010285057 1.31928837 1.33E−05 chr9.0113644978−1.126327 1.33E−05 chr11.0087430751 −0.7455805 1.34E−05 chr2.02379370880.6817732 1.39E−05 chr8.0121030538 0.65272437 1.39E−05 chr19.0050463970−0.6664938 1.39E−05 chrX.0038605033 0.42236218 1.39E−05 chr3.01860791080.83584855 1.42E−05 chr14.0051490944 0.7637686 1.42E−05 chr10.0115370456−0.6105011 1.43E−05 chr5.0153190231 0.74823284 1.44E−05 chr5.00094694140.7091629 1.47E−05 chr22.0030513658 0.64434943 1.47E−05 chr11.00641410751.21208203 1.50E−05 chr6.0125445349 −0.5197668 1.50E−05 chr17.00364017200.69457483 1.55E−05 chr12.0054217430 −0.7282837 1.55E−05 chr5.01683604710.56447978 1.58E−05 chr22.0040175178 0.72636107 1.59E−05chr18.0008153192 0.79448612 1.60E−05 chr14.0104558980 −0.49207841.64E−05 chr2.0045131050 0.61850263 1.69E−05 chr2.0031137180 0.591955851.70E−05 chr14.0023051593 −0.5755742 1.77E−05 chr8.0070251074 −0.59591261.77E−05 chr20.0055924300 0.77477989 1.77E−05 chr12.01328699260.66299989 1.78E−05 chr6.0033974720 0.75180829 1.81E−05 chrX.01184919490.65517163 1.85E−05 chr7.0119364870 −0.5821766 1.87E−05 chr11.0079218833−0.8889979 1.87E−05 chr20.0038615252 1.16961941 1.89E−05 chr6.00223166280.672077 1.94E−05 chr3.0167446192 0.72409551 1.94E−05 chr13.0025744474−0.6265788 1.98E−05 chr18.0008967618 0.64674662 1.99E−05chr12.0057608666 1.18712866 2.02E−05 chrX.0063901420 0.53883304 2.02E−05chr22.0044894647 0.79731823 2.03E−05 chr18.0073417261 0.845274712.05E−05 chr7.0084956359 0.66869985 2.05E−05 chr18.0024532397 0.721428092.05E−05 chr16.0085377991 0.52470179 2.06E−05 chr19.0032252343−0.6132411 2.07E−05 chr18.0037333341 0.72332161 2.10E−05chr15.0062364524 0.81978089 2.10E−05 chr10.0122349007 0.5975229 2.14E−05chr19.0044273158 −0.6421758 2.16E−05 chr6.0162260569 0.7387621 2.16E−05chr20.0001813479 −0.7895873 2.16E−05 chr6.0044598826 0.74785716 2.16E−05chr12.0053690492 0.73301751 2.18E−05 chr7.0114558533 0.66642435 2.19E−05chr10.0027663158 0.50367803 2.21E−05 chr13.0113393004 0.577226422.25E−05 chr12.0088812781 0.73396966 2.26E−05 chr15.00298739950.84952863 2.27E−05 chr3.0077544810 0.7291718 2.28E−05 chr13.00577457220.83463591 2.29E−05 chr13.0094478408 0.61091781 2.33E−05chr13.0110921029 0.73210605 2.35E−05 chr17.0050903002 0.555892112.39E−05 chr7.0118399916 0.56107487 2.41E−05 chr20.0043423337 0.667004412.42E−05 chr12.0060613750 0.70141486 2.42E−05 chr11.00042334810.98622828 2.42E−05 chr18.0065707474 0.67163249 2.43E−05chr17.0042435592 −0.5371504 2.44E−05 chr12.0117663292 0.679959552.46E−05 chr5.0108056803 0.69909423 2.47E−05 chrX.0047639461 0.48051582.47E−05 chr13.0086470118 0.97624718 2.48E−05 chr13.00599512200.62593416 2.52E−05 chr3.0013182915 0.48696429 2.52E−05 chr16.00657796230.66463129 2.57E−05 chr8.0123692428 −0.6304996 2.57E−05 chr2.0083749925−0.621556 2.60E−05 chr2.0031912745 0.5429896 2.64E−05 chr5.01467472490.8371562 2.66E−05 chr5.0034850751 0.65267297 2.68E−05 chr12.01138811540.65084345 2.69E−05 chr20.0031337087 −0.6450485 2.73E−05chr19.0051345820 0.66315546 2.74E−05 chr16.0022628906 1.060322112.74E−05 chr14.0034874938 0.86787504 2.82E−05 chr16.00221623220.50718153 2.82E−05 chr20.0024545582 0.6483386 2.83E−05 chr16.00512399990.76967706 2.83E−05 chr14.0086837080 0.72790847 2.87E−05 chr3.0045730624−1.1348577 2.87E−05 chr8.0011113648 0.54785681 2.89E−05 chr5.00175825862.13401852 2.91E−05 chr4.0125269456 0.4601591 2.94E−05 chr22.0050166610−1.4274981 3.00E−05 chr3.0191599485 0.9379011 3.02E−05 chr12.00134806920.63874404 3.03E−05 chr20.0002645380 1.37839817 3.03E−05 chr4.0154572473−0.762115 3.04E−05 chr14.0096735677 0.79552884 3.06E−05 chr20.00030815080.63818167 3.11E−05 chr11.0093475384 −1.0538671 3.18E−05 chr2.0127944138−0.7316796 3.20E−05 chr16.0077290798 0.71394838 3.22E−05 chr5.01119434830.66157324 3.31E−05 chr14.0105673580 0.60292478 3.33E−05 chr3.00045364741.18245978 3.36E−05 chr17.0008534871 0.84096157 3.37E−05 chr7.00733194130.52199307 3.37E−05 chrX.0102955147 0.41763407 3.37E−05 chr18.00195382120.72580881 3.39E−05 chr6.0052050745 0.62611075 3.42E−05 chr7.01185296640.484139 3.43E−05 chr6.0074871011 0.79821896 3.44E−05 chr20.0049638010−1.179746 3.52E−05 chr5.0011125653 0.56518526 3.52E−05 chr5.00624768760.81475558 3.57E−05 chr2.0142741433 −0.4886771 3.64E−05 chr5.01808573650.58771246 3.65E−05 chr18.0020681043 0.55257366 3.66E−05 chr6.0147480762−0.6741294 3.72E−05 chr5.0151997255 0.59437445 3.73E−05 chr2.02090900400.53083583 3.78E−05 chr13.0036140625 0.8078307 3.78E−05 chr15.00663327690.683103 3.80E−05 chr3.0189770054 0.62026038 3.82E−05 chr12.00205736450.62428502 3.83E−05 chr16.0011480658 −0.6851073 3.84E−05chr20.0047542258 0.86499833 3.86E−05 chr4.0083822472 −0.9056179 3.90E−05chr20.0022843630 −0.7686303 3.95E−05 chr5.0148540678 −0.6035131 4.00E−05chr19.0051551164 −0.7927634 4.10E−05 chr22.0036674237 −0.80483374.11E−05 chr5.0089575203 0.65230132 4.15E−05 chr21.0022014524 0.463179464.20E−05 chr3.0042650476 −0.7421869 4.22E−05 chr19.0007922405 0.511915654.23E−05 chr9.0102164104 0.70823466 4.25E−05 chr3.0054003756 0.740486844.25E−05 chr8.0025546618 0.87526386 4.27E−05 chr12.0000856233 0.727634424.27E−05 chr17.0044984463 −0.5763985 4.29E−05 chr5.0032042842 0.640833314.31E−05 chr8.0033609677 0.61856466 4.32E−05 chr4.0148459836 0.434604444.35E−05 chr6.0020323387 0.65007173 4.36E−05 chr19.0000664734 0.682057594.37E−05 chr6.0029040308 0.6661952 4.38E−05 chr2.0233623357 0.341462134.40E−05 chr6.0026021290 −0.9270936 4.41E−05 chr13.0063032156 −0.75416264.41E−05 chr12.0069480663 0.62097894 4.41E−05 chr17.00213208890.50800652 4.44E−05 chr8.0095024600 0.61617718 4.46E−05 chr21.0028306298−0.4355619 4.53E−05 chr20.0061048321 −0.7294245 4.58E−05chr17.0008482325 0.62234019 4.59E−05 chr20.0004519443 0.656993124.59E−05 chr5.0063380472 −0.5993912 4.60E−05 chr3.0125843470 0.695313814.64E−05 chr20.0044576281 0.6310868 4.65E−05 chr8.0118993090 −0.84258444.65E−05 chr12.0000248545 −1.0664604 4.70E−05 chr14.00694757620.51841343 4.70E−05 chr6.0077620916 0.62696648 4.70E−05 chr11.00833984090.66369636 4.73E−05 chr7.0102248375 0.70170087 4.73E−05 chr18.00431514230.67330864 4.74E−05 chr7.0135468282 0.86163115 4.75E−05 chr5.00435146960.97435005 4.76E−05 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EQUIVALENTS

Although preferred embodiments of the invention have been describedusing specific terms, such description is for illustrative purposesonly, and it is to be understood that changes and variations may be madewithout departing from the spirit or scope of the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

1. A method of determining breast cancer status of a subject, the methodcomprising: determining a methylation state for each of a plurality ofcytosine-guanine dinucleotide (CpG) sites in a sample obtained from thesubject, calculating a cancer presence differential methylation leveland an invasiveness differential methylation level based on themethylation states of the plurality of CpG sites, and comparing thecancer presence differential methylation level and the invasivenessdifferential methylation level to a predetermined cancer statusreference level and a predetermined invasiveness reference level,wherein when the cancer presence differential methylation level deviatesfrom the predetermined cancer status reference level, the presence ofbreast cancer is indicated in the subject, and when the invasivenessdifferential methylation level deviates from the predeterminedinvasiveness reference level, the presence of invasive breast cancer isindicated in the subject.
 2. A method of detecting breast cancer in asubject, the method comprising: determining a methylation state for eachof a plurality of cytosine-guanine dinucleotide (CpG) sites in a sampleobtained from the subject, calculating a cancer status differentialmethylation level based on the methylation states of the plurality ofCpG sites, and comparing the cancer status reference differentialmethylation level to a predetermined reference level, wherein when thecancer status differential methylation level deviates from thepredetermined reference level, the presence of breast cancer isindicated in the subject.
 3. A method of determining if breast cancer ina subject is invasive, or non-invasive, the method comprising:determining a methylation state for each of a plurality ofcytosine-guanine dinucleotide (CpG) sites in a sample obtained from thesubject, calculating an invasiveness differential methylation levelbased on the methylation states of the plurality of CpG sites, andcomparing the invasiveness differential methylation level to apredetermined reference level, wherein when the differential methylationlevel deviates from the predetermined reference level, the breast cancerin the subject is invasive.
 4. The method according to claim 1, whereinthe plurality of CpG sites comprises at least one selected from the CpGsites listed in Table 3 or Table
 15. 5. The method according to claim 3,wherein the plurality of CpG sites comprises at least five selected fromthe CpG sites listed in Table
 21. 6. The method according to claim 1,wherein the plurality of CpG sites comprises at least ten selected fromthe CpG sites listed in Table 3 or Table
 15. 7. The method according toclaim 3, wherein the plurality of CpG sites comprises at least tenselected from the CpG sites listed in Table
 21. 8. The method accordingto claim 1, wherein the plurality of CpG sites comprises at least m %selected from the top n most predictive CpG sites listed in Table 3and/or Table 15, wherein: m is selected from the group consisting of:50, 60, 70, 80, 90, 95, and 99; and n is selected from the groupconsisting of 25, 50, 100, 500 and 1,000.
 9. The method according toclaim 3, wherein the plurality of CpG sites comprises at least m %selected from the top n most predictive CpG sites listed in Table 21,wherein: m is selected from the group consisting of: 50, 60, 70, 80, 90,95, and 99; and n is selected from the group consisting of 25, 50, 100,500 and 1,000.
 10. The method according to claim 1, further comprising:providing treatment for breast cancer to the subject when breast canceris indicated.
 11. The method of claim 8 wherein treatment for breastcancer comprises the administration of medication, radiation or surgery.12. The method according to claim 1, wherein calculating a differentialmethylation level comprises adding in a linear weighted summation valuesbased on the methylation states of the plurality of CpG sites.
 13. Themethod according to claim 1, wherein the sample is a blood sample. 14.The method according to claim 1, wherein the sample is tumor tissue. 15.The method according to claim 1, wherein the subject has or is suspectedto have ductal cell in situ carcinoma.
 16. The method according to claim1, wherein the subject has or is suspected to have triple-negativebreast cancer.
 17. The method according to claim 1, wherein the subjecthas or is suspected to have hormone receptor positive (ER⁺PR⁺) breastcancer.
 18. The method according to claim 1, wherein the subject has oris suspected to have HER2⁺ breast cancer.
 19. The method according toclaim 1, wherein the subject is being monitored for the local orsystemic recurrence of breast cancer.
 20. The method of claim 3, whereinthe plurality of CpG sites comprises at least one selected from the CpGsites listed in Table 27.